.'Ey 0DDE75t.H7D7 HoUingCT Corp. pH 8.5 REPRINT FROM SOIL SCIENCE RUTGERS COLLEGE ASSIMILATION OF ORGANIC NITROGEN BY ZEA MAYS AND THE INFLUENCE OF BACILLUS SUBTILIS ON SUCH ASSIMILATION^ A dissertation submitted in partial f ullillment of the requirements for the degree of Doctor of Philosophy in the University of Michigan By , Reed O. Brigham 1 Publication No. 156, Botanical Department, University of Michigan. Reprint from Soil Science, vol. 3, no. 2, February, 1917. 565 D« Of BJ NOV ! 191f ASSIMILATION OF ORGANIC NITROGEN BY ZEAMAYS AND THE INFLUENCE OF BACILLUS SUBTILIS ON SUCH ASSIMILATION' By Reed O. Brigham, Instructor, University of Cincinnati Statement of Problem The aim of the work presented in this paper was first, to determine whether higher plants can utilize organic nitrogen directly without its be- ing acted upon by microorganisms ; second, to establish the relative im- portance of the compounds used ; and third, to determine how the utiliza- tion of organic compounds by plants is affected by the action of a bac- terium known to be able to decompose such compounds with the produc- tion of ammonia. The work embodies a series of experiments on the in- fluence of different nitrogenous compounds, in sterile and inoculated cul- tures, upon the growth of seedlings of tAVO varieties of Indian corn. The problem was carried out under the direction of Professor J. B. Pollock of the Botany Department of the University of Michigan, and the author wishes here to make grateful acknowledgement to him for his assistance. Historical Introduction The discussion of soil fertility in modern times has centered upon the nitrogen problem. Nitrogen has long been known as one of the elements necessary for plant growth and is the one which must most continually be provided to keep up soil fertility, because it exists in such small quantities in the soil and is so easily removed by crops or by natural processes. As long ago as 1835 Boussingault (5) showed that certain seeds con- tained as high as 4 to 7 per cent of nitrogen calculated on the dry weight basis. Later he (7) grew lupines, beans, and cresses in sand deprived of all nitrogen, and obtained about 1.3 per cent of nitrogen, showing prob- ably a minimum requirement of that element in the plants. In the growth of soil fungi under nitrogen starvation conditions, Goddard (13) obtained from 1 to 2 per cent of nitrogen in the mycelium. The growth of higher plants with an abundant supply of nitrogen shows that element to vary from 4.5 per cent in the leaves of red beets and in peas, 2.3 per cent in wheat grains, to 0.3 pe r cent in rye straw, ac- 1 The data presented in this paper are from a thesis prepared in partial fulfillment of the re- quirements for the degree of Doctor of Philosophy in the University of Michigan. Received for publication October 25, 1916. (155) 156 SOIL SCIENCE cording to Jost (17). With soil fungi grown in a rich nitrogenous medium, Goddard (13) found about 5 per cent in the mycelium. The analyses of different species of mushrooms, as given by Atkinson (1), shows the amount of nitrogen to vary from 2 to 6 per cent. The plant has three possible sources of nitrogen, namely, free nitrogen of the air and inorganic and organic compounds in the soil. The nitro- gen problem has centered around first one and then another of these sources, and in later times about the action of bacteria in relation to all three sources. The view was held from the time of Aristotle to about the end of the eighteenth century that humus was the source of all nourishment of plants, though the early Romans knew that the growing of leguminous crops on the fields in some way increased their fertility and they applied this knowledge to their argriculture. Following the discovery of the chemical elements the relation of these elements to the nutrition of plants became the subject of numerous investigations. The view of Aristotle dominated until about 1840 even though Ingen- houze thought plants were able to absorb free nitrogen from the air. At this time the great German chemist Liebig (26) concluded that plants absorb all or most of their nitrogen in the form of ammonium compounds, that the nitrogen problem was purely chemical, and that free nitrogen could not be utilized. He held firmly to these conclusions throughout his life. Liebig's opinion probably hindered further progress at this time, because he was recognized as one of the greatest chemists and his views were generally accepted. A new view was established about 1860, namely, that nitric acid or nitrates furnish an excellent, if not the most available source of nitrogen for the great majority of plants. This was given by Boussingault (6, 8, 9) at the conclusion of experiments carried on from 1835 to 1860. The problem of the Leguminosae increasing the nitrogen was not ex- plained by these views of Liebig and Boussingault, and numerous experi- ments were carried out, among which were those of Lawes, Gilbert and Pugh (23). These early investigations finally culminated in the experi- ments of Hellriegel and Wilfarth (15). They showed in the^ltlearest way that microorganisms present in the soil are the cause of the formation of the nodules upon the roots of leguminous plants, and that when these nodules are present the assimilation of free nitrogen occurs. These conclusions in regard to bacterial action in the nitrogen prob- lem were followed in a short time by further proof of the role of bac- teria in nitrogen transformation. It was Winogradsky (56) in his bac- teriological studies, who ultimately cleared up the physiology of the nitro- bacteria, and his work has the right to be considered as one of the most important discoveries in plant physiology. He presented conclusive evi- dence of the existence of two kinds of nitrobacteria ; one of which pro- BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 157 duced nitrites from ammonia, and the other nitrates from nitrites. This discovery gave a clearer understanding of the old views of Liebig and Boussingault, and showed how organic compounds can become the source of nitrogen after first being ammonified and nitrified. In this pro- cess organic nitrogen is changed to inorganic which then is available for direct assimilation either as ammonium compounds or nitrates. Wino- gradsky (57, 58, 59) also discovered the non-symbiotic nitrogen-fixing bacteria living in the soil and studied their characteristics. Organic Compounds The direct assimilation of organic nitrogenous compounds was a part of the old humus tlieory and was held until Liebig's chemical theory be- gan to prevail [Meyen (31) 1838], Experiments tending to prove direct assimilation of such compounds were first made in 1857 by Cameron (10), with positive results. About 10 years later Wolf and Knop (60) also did similar work, Baessler (2), Lawes and Gilbert (22), and Berthelot (3) also have done some valuable work along this line. Since in these early experiments the significance of bacteria was not understood and the necessit}^ for pure cultures was not recognized, all these early results are open to question. Recent work on the assimilation of organic nitrogenous compounds has taken into account the possible action of bacteria and various investiga- tions have indicated that these compounds are available for plants, al- though both negative and positive results have been obtained for the same compounds by various investigators. Strictly sterile conditions must be observed in testing accurately whether these compounds are directly as- similable or must first be acted upon by microorganisms to be ammonified or nitrified, or whether when so acted upon, they are rendered less toxic or more fully utilizable. There is also to be considered the difference in availability of the same substance for different plant species. Suzuki (52) found that yellow lupines, potatoes, wheat and Halesia hispidum produced more asparagin from urea than from ammonium salts, while barley did not ; and that, unlike nitrates, urea gave rise to asparagin in etiolated shoots. Pryanishnikov and Lyebyedyev (40) in 1897 carried out experiments in sterilized and non-sterilized media with hippuric acid, urea, leucin, as- paragin and aspartic acid. They found that none of the substances tested approached calcium nitrate as an effective source of nitrogen either in the sterilized or the non-sterilized media; also, that sterilization in all cases reduced the availability of the nitrogen of the organic substances, in most cases no gain being obtained in sterilized media. Nakamura (37) in making quantitative comparison of asparagin and ammonium succinate as sources of nitrogen for barley, onions and Asper- 158 SOIL SCIENCE gillus oryzae, found that, in the case of the phanerogams, fully 50 per cent more growth was made where asparagin was added to the nutrient media than where the other compound was used. This wa% also true in the case of the fungus. In 1898 Lutz (30) carried out some very extensive experiments upon the assimilation of organic nitrogen. These experiments v/ere performed under sterile conditions, and thus fermentation products were excluded and nitrogen fixation prevented. The plants were grown in sterilized sand. The species used were, Cucurbita maxima, Zea mays, Cucumis prophetarum, Helianthus annuus, Ipomaea purpurea, Onicus benedictus, and Cucumis melo. Trimeihylamin, dimethylamin, monomethylamin, diethylamin, propy- lamin and butylamin were all assimilated by the plants without first being fermented in the soil, AUylamin and benzylamin were found to be un- favorable to the growth of phanerogamic plants. The phenol-animes were toxic and the hydramines and pyridin bases were not assimilated. Tetra- methylammonium and tetraethylammonium were not assimilated by phan- erogamic plants. Among the alkaloids he found that caffin and quinin were toxic and cocain, atropin and morphin were not available. Thompson (54) concludes from his studies with oats and barley that lu-ea and uric acid have the same value for the grasses as nitric nitrogen, urea being slightly better than uric acid. His results indicate, however, that hippuric acid is detrimental to plant growth. Pfeffer (39) has found that many heterotrophic organisms either re- quire a supply of peptone or other proteins or attain their maximum de- velopment only when thus supplied. Phanerogams and algae can also em- ploy as more or less valuable sources of nitrogen various organic sub- stcmces such as : urea, glycocoll, asparagin, leucin, tyrosin, guanin, uric acid, acetamid, but none is as favorable to grow^th as sodium nitrate. He has also found that hippuric acid is decomposed by plants into glycocoll and benzoic acid, the latter of which is useless. He believed that the parts of the plant where such decomposition occurs are probably the same as those in which proteins are synthesized. Pfeffer holds that under natural conditions phanerogams rarely absorb organic nitrogenous compounds. Schulze (49) quotes the investigations of a number of experimenters on the assimilation of leucin and tyrosin by plants and describes experi- ments of his own witli lupines, vetches and castor beans, which showed that these chemicals could be used as sources of nitrogen by phanerogams. Sawa (41), from investigations to determine if urea had any action on phanerogams, concluded that urea exercised an injurious action since the control plants made twice the growth of those in the solutions contain- ing urea, and the branches were more vigorous on the control plants. Kawakita (18) in his experiments on the efifect of guanidin on plants found that solutions containing 0.5 gm. of guanidin in 250 c.c. killed BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 159 young barley plants in 3 days and that solutions one-fourth as concen- trated killed the plants in 2 weeks. MoUiard (33) studied the value of asparagin and urea because, as he says, the assimilation of these two substances has been reported by others with different results. He grew his plant under sterile conditions and concludes from his experiments that these two substances maintain a nu- trient role for higher plants. Lefevre (24) in a series of experiments conducted with plants grown without carbon dioxide, found that glycocoll, alanin, tyrosin, and leucin not only furnish nitrogen, but also furnish the carbon required, Schreiner and Reed (44) in their extensive studies tried guanin, al- though it is only slightly soluble in water. They vised it in amounts vary- ing from 1 to 40 parts per million, and in all of these concentrations it bad a slightly beneficial effect upon the growth of wheat plants. Guanidin carbonate, however, when tested on wheat plants in distilled water showed a very strong toxicity. When this solution was treated with carbon black, not only was the toxic action counteracted but the plants gave a better growth than the check in distilled water. In a later publication (45) the same authors in their experiments fotmd guanidin carbonate even in solutions so dilute as one part per million suf- ficient to kill wheat seedlings. Guanin was not harmful. Their experi- ments showed further that for wheat seedlings leucin and asparagin are not at all toxic. Alanin and glycocoll were slightly injurious at higher concentrations. Cumarin was extremely poisonous. Bierema (4) reported that formamid and acetamid were not readily assimilated, although the latter was capable of supplying both nitrogen and carbon. Guanidin carbonate alone was not actively assimilated, but was somewhat more readily taken up in the pressence of calcium lactate, sucrose and glycerol. Uric acid was completely converted into ammonium carbonate, but less readily into urea. Leucin and tyrosin, especially the first, were readily assimilated, ammonium acetate more readily, especially in the presence of dextrose, and ammonium butyrate was still more readily assimilated. Molliard (34) in further researches upon the utilization of organic nitrogen by higher plants, grouped his investigations under three main heads: (a) the action of various organic nitrogenous substances on the development and production of green and dry matter; (b) the total nitro- genous content of plants thus grown, and (c) the formation of protein substances from the absorbed nitrogen. The following substances were used in the culture media in the ratio of 1:1000 parts: urate of sodium, aspartic acid, asparagin (1:500), gly- cocoll, legumin, cyanide of sodium, amygalin, hydrocyanic acid, leucin, tyrosin, myronate of potassium and alanin. Of these substances the first nine were utilized by the plants as shown by the increase in green and 160 SOIL SCIENCE dry matter over similar plants grown as checks. This utilization was the greatest in the case of urate of sodium, and decreased in order named down to leucin. Tyronsin, myronate of potassium and alanin were toxic to the roots only. The amount of protein nitrogen foimd in seedlings grown in the presence of asparagin and glycocoll was about twice the total nitro- gen of the ungerminated seeds. Hutchinson and Miller (16) in their work conclude that, while peptone and certain other nitrogenous compounds may be taken up and to some extent utilized by plants, they are unable to furnish the whole of the nitro- gen required, or at any rate, to supply it with sufficient rapidity. They further conclude that their results are not sufficiently numerous to make it possible to trace any connection between the assimilability or non-assim- ilability of nitrogenous compounds and their constitution. They found it impossible to adhere to their original intention of sterilizing the media, for, although sterile media were most suitable, their employment was pre- vented by the impossibility of sterilizing many of the most desirable sub- stances without more or less decomposition. They grouped the com- pounds experimented with under five heads, namely: (a) readily assimi- lated — ammonium salts, acetamid, urea, barbituric acid (with calcium carbonate), alloxan, humates; (b) assimilated — formamid, glycin, a. aminopropionic acid, guanidin hydrochloride, cyanuric acid, oxaraid, sodium asparatate, peptone; (c) doubtful — trimethylamin (contrary to the results of Lutz), papa-urazine, hexamethylenetetramin ; (d) not as- similated — ethyl nitrate, propionitrile, hydroxy lamin hydrochloride, methyl carbamate; (e) toxic — tetranitromethane. This grouping, they affirm, is applicable only when peas are used and, as the authors suggest, it is possible that other plants may be able to utilize some of the sub- stances which with peas have given negative results. Glycocoll in one culture gave an increase and in another a decrease. Kossowicz (21) in his studies upon the assimilation of guanin and guanidin by mould fungi, found of about 10 fungi experimented with that all were able to utilize guanin as a nitrogen source, also guanidin un- der the conditions favoring the formation of ammonia. It is of interest that these results are different from those with the higher plants. Schreiner (43) in his researches found that when creatin and ni- trates are present less nitrates are used by the plant, although a larger plant growth takes place. The plant absorbs the creatin and builds it into its tissues. The author states that, upon his rather extensive inves- tigations, he is ready to formulate the theory that the degeneration pro- ducts of protein are absorbed directly by the plant from the soil and that the plant uses these units for building up the complex proteins as far as it is possible to do so. Since the plant must spend much energy in the building up of nitrates into amido groups of protein molecules, it is rea- sonable to suppose that the unit part of the complex molecule, when pre- BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 161 sented to the plant, will be used by it in preference to expending labor on the nitrate. The use of these decomposition products gives a different point of view to the problems of soil fertility. Skinner (50) has shown in his experiments that the action of crea- tinin and creatin on growth is very similar. They had a beneficial effect on the growth where nitrate nitrogen was lacking and where only small amounts of nitrate were present, but when large amounts of nitrates were present these compounds produced no effect. Skinner and Beattie (51) report that in all the plants experimented with asparagin is beneficial to growth, even when nitrate is present, al- though to a lesser degree. Schreiner and Skinner (46) report upon some of the nitrogenous soil constituents as follows : Guanin at a concentration of 40 parts per mil- lion showed an increase in growth of 5 per cent over that of the growth in a distilled water control, and good root development. Asparagin showed, both in cultures with and without other sources of nitrogen, a decidedly beneficial effect upon the growth of plants. Guanidin produced a very decided toxic influence on growth. Glycocoll ( amido-acetic acid) in water solutions was found to be beneficial. Alanin, in lower concen- trations, was beneficial to growth, although in concentrations as high as 500 parts per million it slightly injured the roots of wheat seedlings, Dachnowski and Gormley (12) in studies on bog plants and transpira- tion, togetlier with the effect of glycocoll, state that tlie glycocoll is in part undoubtedly the gycocoll absorbed and assimilated. Schreiner and Skinner (47) in experimenting upon the action of methyl glycocoll and glycocoll, found that the first was harmful, and the latter beneficial to the growth of plants. It will be seen from the above review of the work on tliis subject that there is a great deal of contradiction in results obtained by different workers. Bacterial Action The process of nitrification was first shown in 1877 to be dependent upon the presence of certain micoorganisms, by Schloesing and Miintz (42). In 1893 Miintz and Coudon (36) showed for the first time that ammonia production in the soil is due to bacteria. However, in 1862 Pasteur (38) was the first to prove that the formation of ammonia from urea was brought about by the action of mocroorganisms. Within the last twenty years the work of numerous investigators shows that am- monia production from organic nitrogen is a function of most of the soil bacteria. Among the soil bacteria with this capacity is Bacillus subtilis. Miquel (32) shows in his numerous experiments the effect of some of the species of bacilli which play an important role in the ammonifying of urea and splitting of uric acid into urea and other compounds. In (iii— 13^ 162 SOIL SCIENCE his conclusion he suggests that this sphtting may play some part in the availability of these substances for the growth of plants. Lohnis (29) found that soil bacteria rapidly convert urea into ammon- ium carbonate, probably by the action of Urobacillus Pasteurii. The experiments regarding the decomposition of uric acid by bacteria, carried on by Liebert (25) showed that by aerobic bacteria the acid was broken up into carbon dioxide, ammonia, and the intermediate products^, allantoin, urea, and oxalic acid. Lipman (27) has recently determined that B. subtilis changes about 19 per cent of nitrogen present into ammonia. Kelly (19, 20) has recently made extensive studies upon the bio- chemical decomposition of nitrogenous substances and ammonification, using commercial products such as casein, dried blood, cottonseed meal and linseed meal. The results showed that the different materials were converted into ammonia at greatly different rates and amounts. New Experiments In view of the contradictory results found by different investigators on the assimilation of organic nitrogen and in view of the desirability of testing more species of plants for their capacity- of assimilating organic compounds, it was considered worth while to undertake the study of this problem with Mays plants grown with their roots in media free from bacteria except such as were intentionally inoculated into the cultures. The bacterium chosen was B. subtilis. This choice was so made because it is one of the common and widely distributed soil bacteria and has been shown to be capable of ammonifying organic compounds. Methods and Techniqe It is of the greatest importance that sterile, bacteria-free cultures be employed in investigations of soil bacteria, and especially in the case of experiments relative to the availability of organic nitrogen, for only thus can nitrification and ammonification be certainly prevented. It is first necessary to select a medium which may be kept absolutely sterile throughout the experiment and which will permit the plants to make an active growth during a long period. Three media suggest them- selves, namely, sand, water, and agar. Cultures in which each of these was employed were experimented with, and the latter was found to be the best adapted to the work at hand. The plants which were placed in the sand cultures made a very poor growth and seemed to show evidence of a toxic influence. Warington (55) claims that such a material is in several respects a very unnatural medium for plant growth and is generally unsuited for this kind of inves- tigation. Furthermore, it has so low a Avater-holding capacity that a cul- ture when saturated contains about 60 per cent of inert material. Be- cause of the small amount of water, some means of supplying sterile water BRIGHAM—ASSIMILATION OF ORGANIC NITROGEN 163 during the growth must be devised and this adds to the danger of con- tamininating the culture with fungi and bacteria. Water cultures have been found satisfactory for a great deal of work by physiologists, but they require frequent change for the best results, and this is impractical under sterile conditions. Some means of aeration may be used. However, this can be done only at intervals, for contin- uous aeration is not practical with large numbers of cultures. Combes (11) suggests a method of aeration at intervals, but it requires special culture jars not easily obtainable. A preliminary experiment showed the poor growth of Zea plants in non-aerated water cultures. Of the three media mentioned, the agar seemed then to afford the best substratum for the growth of the plants, the chemical compounds contain- ing the mineral matters necessary for plant growth being added to it, of course. Some of the advantages of this media are : it makes possible the most rigidly pure cultures ; the transparency of the agar permits the roots to be at all times visible ; contaminations are easily recognized ; and it af- fords a good mechanical support to the plants. The medium requires no attention beyond the initial preparation, that is, if a sufficient amount of medium is used at the beginning it does not require to be restored or re- newed, even during a long period. This greatly lessens the danger of contamination. A 1-per cent agar solution was employed. This con- tained relatively little inert material, and sufficient water to last several months. The roots of the plants grew largely on the outside of the jelly- like, agar mass, which, as it gradually shrank away from the walls of the vessel, allowed good aeration of the roots. Agar vv^as first employed as a culture substratum for green plants by Harrison and Barlow (14), who made use of it in their experiments with leguminosae. In the culture flasks in the experiment here recorded many of the plants grew well until all the water in the medium had been absorbed and the agar was dried down to a very small mass around the roots. The agar medium was used in all of the experiments herein described, after the first preliminary ones. The roots of the plants were grown un- der sterile or inoculated conditions and the upper part exposed to the air. In order successfully to secure these conditions, some suitable culture jars had to be provided. For this purpose Erlenmeyer flasks of Jena, Resistenz or Bohemian glass of 700 and 1000-c.c. capacity were used. These were nearly filled with agar medium and sterilized in the autoclave for 20 minutes at 12 to 15 pounds pressure. Following the methods of Hutchinson and Miller (16), Schulow (48) and Combes (11) cotton plugs were placed in the mouths of the flasks, each plug rolled around a glass tube about 1 cm. in diameter and 15 cm. in length, through which the young plant could grow. This tube was also plugged with cotton. When the top was reached by the plant, the tube was withdrawn and the cotton pressed about the plant. This method allowed free growth of the leaves in the air, and aeration as well as sterile condition of the roots. 164 SOIL SCIENCE where this was desired. Each culture flask was wrapped with black paper to exclude light from the roots. The experiments described here were carried out with two varieties of com, namely, Zea Mays everta, Sturtevant (pop corn) ; and Zea Mays indentata, Sturt. (dent corn). The pop corn seedlings were all grown in the greenhouse the first year, but the dent corn seedlings the second year were grown in the large south windows (9 feet high and 12 feet wide) of the laboratories of the Science Building, because the new botanical greenhouses were not completed and the old one was unavailable. The air of the rooms was kept moist by sprinkling the floors and having large pans of water exposed in the room. The cultures were maintained for two to three months. Two methods were employed for measuring the growth of the plants. During the growth at various intervals and at tlie completion of the ex- periment the leaves were measured, and with the dent corn the dry weight of both the tops and the roots was obtained. The measurements were made first from the seed to the top of the youngest leaf and to this was added the length of each leaf from the stalk to its tip. The complete mea- surement of the plant was recorded. The measurements at intervals dur- ing growth did not reveal any special characteristics so they are not re- corded in the data presented in this paper. The dry weights of the whole plant were determined after the removal of the remains of the seed. The roots were freed from the agar which remained about tliem by melt- ing the agar in the autoclave and then washing the roots in boiling water. All of the substances occurring in the roots which are soluble in hot water were, of course, lost during this treatment. Each plant was then placed in a separate envelope and dried at a temperature of 80° C. to con- stant weight. These data of measurements and weights allow for accur- ate comparison with the checks which were grown in each series. The results were recorded and studied in three different forms : by means of the tables compiled from the figures obtained and recorded later in this paper ; by a comparison of photographs taken of the different sets grown at different times; and by graphs drawn for an easier and more ready comparison. Inoculated and sterile cultures were compared. Twelve cultures were prepared in a set, each containing the same nitrogen source. Six of each set of 12 cultures were sterile and 6 were inoculated. One healthy seedling was planted in each flask. For the first series, the flasks were inoculated with 10 drops of soil water, prepared by shaking 5 gm. of soil with 50 c.c. of distilled water. In all of the other series where the flasks were inoculated, a pure culture of B. subtilis was used. A loop of bacteria was transferred with a sterile platinum wire loop from an agar slant to the warm liquid agar culture flask and thor- oughly distributed through the medium by stirring with the sterile needle and shaking. In every case the inoculated flask showed a good growth of the bacteria. BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 165 Seed Sterilization At the beginning- of the work the seeds were steriHzed by immersing in a water solution of mercuric chloride, 1:500, for 20 minutes. The seeds were first immersed in alcohol to remove any film of air. After the mercuric chloride treatment they were rinsed in sterile water to remove the sterilizing agent. This method was successful for the dent corn ; but when the pop corn was so treated only a poor germination was obtained, and weak seedlings resulted from the few seeds that did germinate. These results made it necessary to^ employ some other method for sterilizing pop corn, and following the suggestion of Lipman and Fowler (28), sulfuric acid (1.84 specific gravity) was tried. The best results were obtained by immersing the seeds for 4 minutes and then rinsing them in sterile water. The sterilized seeds were placed on moist filter paper in sterile Petri dishes and allowed to germinate. In three or four days those that germin- ated well were transferred with sterile forceps to the surface of the agar medium in the flasks, and later the young shoots were directed into the glass tubes, w^hich reached above the cotton stoppers. Germinating the seeds on agar was tried but the surface was too dry for the best results. Great care was used in selecting the seedlings to have them as nearly alike as possible, yet in spite of this precaution there was considerable differ- ence in the rapidity of the growth during the first two weeks. Some that appeared healthy would not reach the tops of the tubes for a week or more after others which seemed equally as good. This difference is one of the greatest sources of error in the method used but its effect is mini- mized by the use of large numbers of plants. Nutrient Solutions The nutrient solution in these cultures was one which has been found to be most successful by Professor Pollock, after extensive experiments in his laboratory. The tribasic calcium phosphate was used instead of the acid phosphate to assure an alkaline medium. This has low solubility but by using an excess of the phosphate the solution was constantly kept sup- plied witli a quantity sufficient for the growth of the plants. The amoimt of the different organic nitrogenous compounds to be used in the various solutions was determined upon the basis of furnishing in each solution the same amount of nitrogen that was present in the .004 M. solution of sod- ium nitrate used. Since peptone does not have a definite chemical for- mula, the nitrogen could not be accurately calculated, but it was esti- mated that 0.2 gm. of peptone in a liter of water would give the required amount of nitrogen. A stock solution was used for the check and to this was added single nitrogenous substances in the preparation of the other media. The follow- ing is a list of the substances used in the stock solution^ together with the 166 SOIL SCIENCE number of grams per liter of water of each substance used : calcium phos- phate (tribasic) 1.240; magnesium sulfate 0.246; potassium chloride 0.298; ferric chloride 5 c.c. of .001 M. solution. This solution furnishes all of the elements necessary for the growth of green plants except nitro- gen, and those obtained from water and carbon dioxide. The other culture media were made up by adding each of the following substances to the stock solution (the number of grams of each used per liter of stock solution is indicated) : sodium nitrate 0.340; urea 0.120 peptone 0.2; guanin 0.120; guanidin carbonate 0.180; benzamid 0.484 caffein 0.194; alanin 0.364; ammonium sulfate 0.264; asparagin 0.264 glycocoll 0.300; uric acid 0.168; diphenylamin 0.676; guanidin nitrate 0.122; hemoglobin 0.634; casein 0.459; linseed meal 1.120; cottonseed meal 1.090; malt 1.596; creatin 0.174. With the exceptions of cottonseed meal, malt, peptone and linseed meal, all of the substances used in these nutrient solutions were chemically pure, and distilled water from the chemical laboratory was used in all cultures. The organic nitrogenous compounds employed were those prepared by C. A. F. Kahlbaum. The cottonseed meal and linseed meal were secured from a retail feed store, and the malt which was obtained from a brewery consisted of ground, sprouted barley grains. Experiments Pop Corn Series I The plants of this series were started the middle of October, 1914, and were harvested the middle of March, 1915. They were grown in the greenhouse in the following media : check consisting of the stock solution ; and separate sets of media compound of stock solution to which sodium nitrate, urea, peptone, guanin and guanidin carbonate were respectively added. Half of the flasks were kept sterile while the other half were in- oculated with soil water, as has been described above. Soil water was not again used for inoculation because of bad results. The addition of this mixed culture of bacteria and fungi from the soil included some parasitic forms, which were detrimental to the plants. Therefore, in the succeeding series a pure culture of B. suhtilis was used. The results of this series are incorporated in Tables I and II. Series II The cultures of Series II were started February, 1915, and harvested in June of the same year. They were grown in the greenhouse, and the same media were used as in Series I. The flasks which were here inocu- lated, had pure cultures of B. suhtilis added, as has been described in the section on methods and technique. The plants of this series made a better and more uniform growth than those of Series I. Some of the plants BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN TABLE I SERIES I, POP CORN, STERILE CULTURES (October, 1914 — March, 1915) 167 Culture Number of plants Total length of leaves cm. Average length of leaves cm. Per cent of average length of leaves of check Check Sodium Nitrate Urea Peptone 4 4 3 4 2 255 200 ISO 145 425 365 335 245 300 290 165 335 315 225 220 180 115 187.50 342.50 251.70 273.75 147.50 100.0 182.6 134.2 146.0 78.6 Guanidin Carbonate*.. Plants small and died within a few days. TABLE II SERIES I, POP CORN, INOCULATED WITH SOIL WATER (October, 1914 — March, 1915) Number Total length Average length Per cent of Culture of of leaves of leaves average length of plants cm. cm. leaves of check Check 5 320 275 250 190 170 241.00 100.0 Sodium Nitrate 4 280 260 230 220 247.50 127.6 Urea 4 575 375 330 300 395.00 163.9 Peptone 4 230 211.25 87.6 220 215 180 Guanin 3 320 290 280 303.00 125.8 Guanidin Carbonate*. . * Plants small and died within a few days. 168 SOIL SCIENCE showed contaminations which were parasitic. These were discarded from the data, and this was done in ail of the following series. The contamina- tions in tlie flasks may have occurred on the seeds, some bacteria or fungi havii-^g survived the seed sterilization, or they may have gained entrance at the time of planting the seeds, when it was necessary to open the flasks. The results obtained here are similar to those of Series I, and may be studied by referring to Tables III and IV, TABLE III SERIES II, POP CORN, STERILE CULTURES (February-June, 1915) Number Total length Average length Per cent of Culture of of leaves of leaves average length of plants cm. cm. leaves of check Check 12 290 235 100.0 285 275 265 250 230 230 210 205 200 195 185 Sodium Nitrate 7 450 445 405 370 355 335 325 384 163.4 Urea 8 370 360 355 295 280 270 230 230 299 127.2 Peptone 10 325 253 107.6 305 290 275 250 230 230 200 Guanin* ' Guanidin Carbonate* . . * Plants small, no roots, and soon died. Series III The plants in Series III were started the last of June, 1915, and har- vested in about 8 weeks, the growth being very rapid during the long days and intense heat of the summer months. The greenhouse had rather poor means of ventilation and the glass was not painted so that the tern- BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 169 perature often reached 52° C, but the plants survived and made fairly good growth. The following nitrogen compounds were used in this set in addition to the check : sodium nitrate, urea, and peptone. The results are similar to those of the preceding series, and are recorded in Tables V and VI. TABLE IV SERIES II, POP CORN, INOCULATED WITH B. SUBTILIS (February-June, 1915) Number Total length Average length Per cent of Culture of of leaves of leaves average length of plants cm. cm. leaves of check Check 11 365 320 246 100.0 315 290 230 225 225 220 190 165 155 Sodium Nitrate 7 610 530 400 400 310 300 ■300 407 165.4 Urea 11 400 390 380 375 370 370 370 365 360 355 335 370 150.4 Peptone 10 440 420 360 320 315 315 310 305 265 250 330 134.1 Guanin* Guanidin Carbonate* . . * Plants small, no roots, and soon died. The study of the data upon these first three series revealed very similar results in all. A summary of these results is shown in Table VII and figure 1. Because of the large number of plants grown, some definite con- clusions may be drawn from the experiments, which will be stated later. 170 SOIL SCIENCE Series IV The plants of this series were grown at the same time as, and under similar conditions to those of Series III, except that water cultures were used instead of agar cultures. There was no provision made for aeration. TABLE V SERIES III, POP CORN, STERILE CULTURES (June- August, 1915) Number Total length Average length Percent of Culture of of leaves of leaves average length of plants cm. cm. leaves of check Check 5 290 210 190 180 180 210 100.0 Sodium Nitrate 3 320 300 220 283 134.7 Urea 3 180 178 178 84.7 175 Peptone ^ 5 420 370 313 149.0 300 1245 230 In addition to the check, media containing the following nitrogen com- pounds were used : sodium nitrate, urea, peptone, benzamid, caffein, alanin, ammonium sulfate, and asparagin. By consulting Tables VIII TABLE VI SERIES III, POP CORN, INOCULATED WITH B. SUBTILIS (June-August, 1915) Culture Number of plants Total length of leaves cm. Average length of leaves cm. Per cent of average length of leaves of check Check 6 4 4 2 320 260 250 230 195 190 430 410 310 170 585 430 325 280 340 260 224 340 405 300 100.0 Sodium Nitrate Urea 151.7 180.8 133.9 and IX and comparing the growth with that in the agar medium, it can readily be seen that the growth in these water cultures was exceedingly poor in all cases, and not nearly equal to that in the agar cultures. The BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 171 TABLE VII SUMMARY OF DATA OF LENGTHS OF LEAVES OF 110 POP CORN PLANTS GROWN IN DIFFERENT CULTURE MEDIA, UNDER STERILE CONDITIONS AND INOCULATED WITH B. SUBTILIS, 1914-1915 Sterile Cultures Inoc. B. subtilis Culture Average length cm. Per cent of average length of check Average length cm. Per cent of average length of check Check Sodium Nitrate. . Urea Peptone Guanin* Guanidin Carbonate* . . . 227.7 353.5 265.7 272.3 100.0 155.2 116.6 119.6 243.9 379.0 379.3 325.0 100.0 159.4 159.5 133.2 Toxic, no growth. POP CORI M cm. 400 300 \ ^-^. s *§*>»» (w! B -» ^ ' ■ tarea —_ Inoc. B.5d zoo \ 100 000 ^ o 1 ft Z5 o zS Fig. 1. — A summary of the results obtained from all of the pop corn plants grown in the experiment. 172 SOIL SCIENCE plants were weak and sickly. It was decided therefore, that unaerated water cultures were unsuitable for these experiments, and thereafter only agar cultures were used. The relative value of the nitrogenous substances in the water cultures was similar to that of these substances in the agar cultures, and may be used in connection with them. TABLE VIII SERIES IV, POP CORN, WATER CULTURES, STERILE CONDITIONS (June-August, 1915) Number Total length Average length Per cent of Culture of of leaves of leaves average length of plants cm. cm. leaves of check Check 4 160 120 110 100 123.0 100.0 Sodium Nitrate 5 310 240 240 190 160 228.0 185.3 Urea 3 165 160 160.0 130.0 155 Peptone 4 230 210 120 170.0 138.0 Benzamid* 2 120 160 140.0 Caffein 113.8 120 Alanin ^ 4 140 140 127.5 103.6 120 110 Ammonium Sulfate . . > 3 160 120 120 133.3 108.3 Asparagin 5 225 120 135.0 109.8 115 110 110 * Toxic, no growth. Dent Corn In the preceeding experiments some difficulty had been found in ob- taining pop com seedlings, and because of this fact and because it was ad- visable to try the effect of these substances upon another variety of the species, dent corn was used. Also other chemical substances were used as sources of nitrogen. Series V The plants of Series V were started in October, 1915, and harvested the following February. The plants were grown in the south window of one of the botanical laboratories. The light was not as good here as in BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 173 the greenhouse, but fairly uniform growth was obtained. The following nitrogen compounds were tested : sodium nitrate, urea, peptone, guanin, guanidin cabornate, guanidin nitrate, benzamid, caffein, alanin, ammon- ium sulfate, asparagin, glycocoll, uric acid, diphenylamine. In these, as in all the experiments, plants were grown in the stock solution as a check. The results of these experiments are given in Tables X and XI. It is interesting to compare these results with those found in the growth of the pop corn plants. In general they are similar, but guanin which was toxic to pop corn was found to be quite beneficial to the dent corn seedlings. TABLE IX SERIES IV, POP CORN, WATER CULTURES, INOCULATED WITH B. SUBTILIS (June- August, 1915) Number Total length Average length Per cent of Culture of of leaves of leaves average length of plants cm. cm. leaves of check Check 5 140 135 135 120 100 126 100.0 Sodium Nitrate 5 185 175 170 170 150 150 166 131.7 Urea 1 120 120 95.2 Benzamidt Caffein S 150 115 110 95 90 112 88.8 Alanin , 2 130 110 120 95.2 Ammonium Sulfate . . , 5 250 160 90 85 80 133 105.5 Asparagin 1 140 140 119.0 No plants obtained. t Toxic, no growth. Series VI The plants of this series were started November, 1915, and the growth terminated during the next March. They were grown in a very poorly lighted window of one of the botanical laboratories, and consequently the growth was poor and very irregular. These facts must be considered in drawing any conclusion from the results of this series. The following nitrogenous substances were used : hemoglobin, casein, linseed meal, cot- tonseed meal, and malt. The results may be studied in Tables XII and XIII. 174 SOIL SCIENCE TABLE X SERIES V, DENT CORN, STERILE CULTURES (October, 1915~February, 1916) Number Totallgth. Avg. length Dry Average Per cent of Culture of « of leaves of leaves weight dry weight av. dry wgt. Plants cm. cm. gm. gm. of check Check 5 285 250 230 180 150 219 0.42 0.28 0.30 0.25 0.25 0.30 100.0 Sodium Nitrate 4 410 325 280 270 321 1.08 0.62 0.63 0.52 0.71 236.6 Urea 4 320 180 160 110 182 0.75 0.30 0.22 0.06 0.33 110.0 Peptone 4 345 296 1.18 0.66 220.0 345 0.60 260 0.50 235 0.35 Guanin 5 404 385 365 350 250 351 1.24 1.40 1.10 1.30 0.46 1.10 366.6 Guanidin Carbonate . 4 165 155 120 105 138 0.35 0.30 0.15 0.20 0.25 83.3 Benzamid* Caffein 6 150 145 135 110 80 25 109 0.20 0.31 0.21 0.20 0.13 0.10 0.19 63.3 Alanin 6 420 365 320 270 225 215 300 1.90 1.28 1.05 0.51 0.47 0.44 .94 313.3 Ammonium Sulfate. . . 3 300 290 235 275 0.65 0.62 0.35 .54 180.0 Asparafifin ....••>■•• 5 365 319 1.40 .84 290.0 335 0.94 315 0.71 310 0.70 270 0.45 Glycocoll 5 365 281 1.14 .63 210.0 325 0.70 245 0.52 240 0.40 230 0.40 Uric Acid • ...• 6 280 270 256 0.48 0.47 .44 146.6 265 0.57 265 0.38 260 0.45 200 0.31 BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN TABLE X— Continued 175 Number Total Igth. Avg. length Dry Average Percent of Culture of of leaves of leaves weight dry vifeight av. dry wgt. Plants cm. cm. gm. gm. of check Diphenylamin* Guanidin Nitrate 4 160 135 130 110 134 0.31 0.27 0.22 0.15 .24 80.0 * No growth, died within three days. Series VII This was one of the most successful sets grown during the year. The plants were started in January, 1916, and harvested in about three months. They were grown in a well lighted window of one of the labora- tories of the Science Building, and the room was well heated. The fol- lowing media were used : distilled water, check, sodium nitrate, urea, pep- tone, guanin, alanin, ammonium sulfate, asparagin, uric acid, hemoglobin, casein, linseed meal, cottonseed meal, malt, and creatin. The plants of this series all made good growth and some interesting results were obtained, which may be readily seen by a study of Tables XIV and XV. The results of this and of the other series are discussed later in this paper. Series VIII The plants of this series were started in February, 1916, and harvested about two months later. They were grown in large test tvibes. These plants had only about half the amount of medium that the plants of other series had, and consequently the growth had to be terminated at an earlier stage, but nevertheless, interesting results were obtained. The plants were grown in the new botanical greenhouse under very ideal conditions of light and heat, and a very good and uniform growth resulted. The following substances were used : check, sodium nitrate, urea, uric acid, casein, and cottonseed meal. The detailed results of this series are given in Tables XVI and XVII. Discussion The data presented in this thesis, comprise observations upon 614 Zea Mays plants grown until the water supply became exhausted in one or more of the culture flasks of a series. The conclusions are based upon the results of growth of these plants. This number does not include those plants which showed extreme toxic effects when young and made no further growth, nor those discarded because they were attacked by fungi. The large number of the plants employed makes it possible for us to draw certain fairly definite conclusions from the data secured. The percentage of possible error in such work is a large one and must be taken into account in interpreting the result obtained. There are sev- 176 SOIL SCIENCE TABLE XI SERIES V, DENT CORN, INOCULATED WITH B. SUBTILIS (October, 1915— February, 1916) Number Total Igth. Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry weight av. dry wgt Plants cm. cm. gm. gm. of check Check 6 215 200 190 175 170 135 181 0.30 0.28 0.30 0.30 0.20 0.12 0.25 100.0 Sodium Nitrate 4 340 270 250 200 265 0.65 0.45 0.38 0.25 0.40 160.0 U rea 5 345 231 0.60 0.30 156.0 370 0.70 165 0.28 160 0.16 145 0.20 Peptone 4 375 345 1.00 0.96 384.0 350 0.93 330 0.70 325 1.20 Guanin 6 400 335 320 275 270 270 311 1.07 1.04 0.47 0.60 0.70 0.50 0.73 292.0 Guanidin Carbonate. . 4 210 175 170 130 171 0.44 0.30 0.25 0.25 0.31 124.0 Benzamid* 6 210 130 0.34 0.23 Caffein 92.0 170 0.23 140 0.22 115 0.22 80 0.20 55 0.15 Alanin 6 430 330 300 1.20 1.50 0.88 352.0 330 1.05 270 255 185 0.55 0.69 0.32 Ammonium Sulfate . . 5 430 345 325 300 300 340 1.52 0.97 0.75 0.65 0.54 0.85 348.0 Asparagin 5 420 400 300 285 275 336 0.90 1.34 0.69 0.68 0.57 0.83 332.0 Glycocoll 5 325 225 220 220 200 240 0.82 0.40 0.67 0.53 0.37 0.56 224.0 BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 177 TABLE XI — Continued Number Total Igth. Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry weight av. dry wgt Plants cm. cm. gm. gm. of check 5 415 320 230 1.02 0.50 0.48 192.0 215 0.32 170 0.29 170 0.27 Diphenylamin* Guanidin Nitrate .... 3 150 130 135 0.26 0.22 0.22 88.0 125 0.20 * No growth, died within three days. eral sources of error of which the most serious is the individual differ- ences which occur between plants. No two individuals are exactly alike, as is shown, for instance, by the different growth vigor of different plants under identical external conditions. The degree of this error diminishes with increase in the number of plants. A second factor is the light rela- tion. In the climate of southern Michigan, during the winter months on TABLE XII SERIES VI, DENT CORN, STERILE CULTURES (November, 1915— March, 1916) Number Total Igth, Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry weight av. dry wgt Plants cm. cm. gm. gm. of check Check 6 330 320 300 300 285 220 289 1.25 1.52 1.22 0.90 1.00 0.55 1.07 100.0 Hemoglobin 5 335 335 280 255 210 283 2.20 0.55 1.34 0.60 0.40 1.02 95.3 Casein 4 400 255 250 230 284 3.00 1.00 0.50 1.35 1.46 130.4 Linseed Meal 5 355 330 315 285 280 313 2.26 1.70 1.05 1.30 1.00 1.46 136.4 Cottonseed Meal 5 405 370 350 310 255 338 1.34 2.20 1.65 1.67 1.55 1.68 157.0 Malt 6 390 380 328 1.25 0.95 1.13 105.6 335 2.35 300 0.85 300 0.75 265 0.65 (iii— 14) 178 SOIL SCIENCE account of the shorter days, less intensity of the sunlight and the large proportion of the cloudy days, the light at the disposal of the plants is much less than during the summer months. As a result, the rate of growth is less than in summer. This fact must be taken into consideration when comparing the growth of series which were grown at different times of the year. Also, those plants which were grown in the laboratory win- dows did not receive as much light as those in the greenhouse, and those standing near the windows received more than plants farther back. This TABLE XIII SERIES VI, DENT CORN, INOCULATED V/ITH B. SUBTILIS (November, 1915 — March, 1916) Number Total Igth. Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry weight av. dry wgt Plants cm. cm. gm. gm. of check Check 5 360 335 325 310 255 316 1.65 I.IS 1.40 0.70 1.40 1.22 100.0 Hemoglobin 4 350 260 260 185 264 0.60 1.30 0.85 0.35 0.78 62.3 Casein 3 400 300 210 303 2.70 1.42 0.50 1.54 126.2 Linseed Meal 4 370 365 315 280 332 2.82 1.30 0.90 2.19 1.61 123.7 Cottonseed Meal .... 6 365 310 300 280 255 230 290 2.98 1.50 1.70 1.17 1.25 1.05 1.61 123.7 Malt S 355 345 340 320 235 310 0.90 1.45 1.25 0.90 0.56 1.01 82.7 was controlled by shifting their positions during the period of growth. The diminishing of light causes a lessening of carbohydrate production and hence slower growth. This slow development may somewhat influ- ence the assimilation of nitrogen. Another possible source of error is the wide temperature variations which occurred while some of the series were being grown. During vacations the heat in the building where the plants were grown was reduced and this caused a check in growth in Series V and VI from which the plants did not fully recover. These factors, then, which influence the percentage of error must be born in mind when mak- ing comparisons between different series. The large error due to differ- ences in individual plants is well illustrated in the check solution of Series BRIGHAM-ASSIMILATION OF ORGANIC NITROGEN 179 TABLE XIV SERIES VII, DENT CORN, STERILE CULTURES (January-March, 1916) Number Total Igth. Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry weight av. dry wgt Plants cm. cm. gm. gm. of check Distilled Water 3 275 215 170 220 0.93 0.55 0.40 0.62 47.3 Check 6 375 360 345 330 305 230 324 1.65 1.20 1.30 1.20 1.41 1.15 1.31 100.0 Sodium Nitrate 4 400 385 345 290 370 2.75 2.60 1.90 1.42 2.17 185.6 Urea 5 465 375 3.12 2.00 152.6 430 2.40 390 1.73 320 1.50 270 1.27 Peptone 5 435 353 2.47 1.78 135.8 365 1.47 350 2.60 310 1.05 305 1.30 Guanin 6 400 360 360 350 345 275 348 2.15 1.90 1.90 1.20 1.42 1.05 1.60 122.1 Alanin 4 335 291 2.00 1.50 114.5 300 2.45 280 0.90 250 0.67 Ammonium Sulfate. . . 6 495 485 460 400 360 350 425 2.70 3.85 3.30 2.02 1.70 1.12 2.45 187.0 Asparagin 6 560 523 5.10 4.25 3.89 296.9 550 550 4.20 510 2.77 500 4.10 470 2.97 Uric Acid 4 560 520 435 400 478 3.85 3.35 2.47 2.98 3.16 241.2 Hemoglobin 6 485 425 365 325 300 285 364 3.75 2.60 1.42 1.20 1.52 1.02 1.92 146.5 180 SOIL SCIENCE TABLE XIV— Continued Number Total Igth. Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry weight av. dry wgt. Plants cm. cm. gm. gm. of check Casein 6 490 460 440 420 400 320 422 3.65 2.54 2.80 3.00 2.70 1.25 2.68 204.5 Linseed Meal 5 415 335 310 300 275 327 2.75 1.35 0.97 1.30 1.50 1.57 112.2 Cottonseed Meal 6 410 400 360 280 245 230 321 2.90 2.15 2.10 1.40 1.07 1.37 1.83 139.6 Malt 6 330 325 320 310 275 250 301 1.25 1.25 1.47 0.97 0.77 0.65 1.05 80.1 Creatin 6 400 360 355 350 345 275 347 2.20 1.45 1.65 1.40 1.45 1.37 1.58 120.6 II. The largest plant measured 365 cm. and the smallest 155 cm., a dif- ference of 210 cm. However, in all the checks of all the series, 48 plants in sterile cultures averaged 227.7 cm., and 49 plants in inoculated cultures averaged 221.6 cm. a difference of only 6.1 cm. With this num- ber of plants the margin of error is very small. The means for determining the amount of development of the plants in the various compounds used was, as has been stated above, by measure- ment of the length of the stalks and leaves, and by determining the dry weight. A comparison of the data obtained by the two methods shows that they are nearly parallel. The data show that in 19 cases the measure- ments and weights are, respectively, in the same relation in the sterile and the inoculated cultures ; but in 5 cases they are reversed. This may be partly explained by the fact that the cultures in which these reverses oc- curred were checked in their growth, as has been explained. The leaves then did not develop well, but ears were formed which increased the weight. The weights probably serve a more definite and accurate basis for comparison than the measurements (cf. Tables X-XVII). Since the problem was to determine the availability of various organic nitrogenous compounds for higher plants, the most logical means of dis- cussion seems to be to take up each compound separately, explain the re- BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 181 TABLE XV SERIES VII, DENT CORN, INOCULATED WITH B. SUBTILIS (January-March, 1916) Number Total Igth. Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry weight av. dry wgt. Plants cm. cm. gm. gm. of check Distilled Water 2 220 140 180 0.72 0.28 0.50 34.5 Check S 350 330 325 265 215 297 1.79 1.68 1.65 1.20 0.95 1.45 100.0 Sodium Nitrate 4 410 370 320 300 350 2.32 2.80 0.80 0.70 1.65 113.8 Urea S 480 465 405 355 225 350 2.35 3.95 1.67 1.65 0.70 2.10 144.8 Peptone 6 490 440 435 3.45 2.32 2.35 162.0 440 1.57 430 3.00 430 1.70 380 2.10 Guanin 6 495 490 470 465 420 385 454 3.55 3.10 2.25 2.35 1.92 1.97 2.52 173.8 Alanin 6 400 400 315 305 300 280 333 2.97 2.95 2.60 1.30 1.40 1.38 2.10 144 8 Ammonium Sulfate. . . 6 510 465 465 460 445 430 462 3.45 3.57 3.17 2.17 3.15 3.41 3.15 217.2 Asparagin 4 610 600 535 500 561 4.69 4.55 3.77 3.95 4.24 292.4 Uric Acid 6 480 475 417 3.32 2.52 2.51 173.1 460 3.55 400 3.12 390 1.60 300 0.97 Hemosrlobin 6 550 502 4.30 3.61 248.0 540 3.15 535 4.27 • 510 3.80 505 3.65 375 2.49 ia2 SOIL SCIENCE TABLE XV— Continued Number Totallgth. Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry weight av. dry wgt Plants cm. cm. gm. gm. of check Casein 4 485 470 464 4.85 3.42 3.79 261.4 450 3.57 450 3.35 Linseed Meal 6 425 375 350 350 330 330 360 3.10 1.65 1.95 1.32 2.85 1.60 2.08 143.4 Cottonseed Meal .... 6 460 400 370 370 360 315 380 3.20 2.82 2.20 2.12 2.52 2.70 2.59 178.6 Malt 6 380 331 1.68 1.55 1.50 103.4 1 375 370 1.75 360 1.92 270 1.10 230 1.02 Creatin 6 390 340 1 .60 1.44 99.3 360 1.45 340 1.20 325 1.72 320 1.17 300 1.50 suits obtained in the different cultures, and show the significance which they seem to reveal. Therefore, this procedure has been adopted. Checks The check solution was used in all the series. It contained all the chemical elements necessary for the growth of plants except nitrogen and those which the plant gets from the air. The growth in this solution was taken as the amount of growth allowed by the nitrogen supply in the seed. In the culture solutions containing nitrogen a growth markedly less than that of the check was interpreted as a toxic effect. A growth equal to that of the check was assumed to indicate that the nitrogen was not avail- able. A growth markedly better than the check indicated that nitrogen in the form supplied was available. The plants grown in the check solu- tion, toward the end of the period of growth, always showed the yellow- ing of the leaves, a characteristic effect of the lack of nitrogen. The difference between the plants grown in the sterile cultures and in the inoculated ones is very slight. With the pop corn the plants inocu- lated in all cases were from 10 to 40 cm. better. Considering the mea- surement on length of all the check plants, those in the sterile cultures averaged 6 cm. per plant better than those in the inoculated. BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 183 Sodium Nitrate A complete nutrient solution containing sodium nitrate was employed in all the series but one. Since the time of Boussingault (8) sodium nitrate has been considered one of the best, if not the best, source of nitrogen. It is in common use as a commercial fertilizer. In Series II, III, and VIII of the experiments it produced the best growth of all sub- stances used. These were all grown in the greenhouse under favorable TABLE XVI SERIES VIII, DENT CORN, STERILE CULTURES (February-April, 1916) Number Total Igth. Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry wreight av. dry wgt Plants cm. cm. gm. gm. of check Check 6 170 170 160 155 ISO 140 157.5 1.52 1.25 1.10 1.30 1.25 1.00 1.23 100.0 Sodium Nitrate 5 215 215 205 200 185 204.0 1.90 1.80 1.72 1.60 1.80 1.76 143.0 Urea 3 190 190 165 181.6 1.85 1.70 1.50 1.68 136.5 Uric Acid 6 200 190 190 185 160 150 179.0 1.80 1.70 l.SO 1.50 1.40 1.45 1.50 126.8 Casein 6 205 200 190 175 170 170 185.0 1.65 1.45 1.70 1.25 1.25 1.20 1.41 114.6 Cottonseed Meal .... 5 160 155 145 145 135 148.0 1.28 1.20 1.10 1.10 1.02 1.14 92.6 conditions of temperature and light. In Series VIII, the experiment was discontinued after only two months had elapsed because with the small amount of medium used the water was exhausted at the end of that period. Table VII and figure 1 show that in the growth of pop corn, sodium nitrate in sterile cultures was the best of the compounds tested as a source of nitrogen, while in the inoculated cultures urea equaled it in value. In the growth of dent corn the results in sterile cultures indicated that am- monium sulfate and asparagin are superior to sodium nitrate as a source 184 SOIL SCIENCE of nitrogen. In inoculated cultures, however, the following substances gave better results than the nitrate : asparagin, ammonium sulfate, pep- tone, guanin, uric acid, alanin, urea, hemoglobin, casein, linseed and cot- tonseed meals. The growth of the dent corn plants in the inoculated cul- tures of the nitrate was slightly poorer than in the sterile cultures, while TABLE XVII SERIES VIII, DENT CORN, INOCULATED WITH B. SUBTILIS (February-April, 1916) Number Total Igth. Avg. length Dry Average Per cent of Culture of of leaves of leaves weight dry weight av. dry wgt. Plants cm. cm. gm. gm. of check Check 6 155 155 140 130 125 120 137.5 1.20 1.15 1.15 0.80 0.92 0.95 1.03 100.0 Sodiiun Nitrate 6 205 205 205 195 190 170 195.0 2.15 1.82 1.85 1.80 2.10 1.55 1.88 182.5 Urea 6 180 175 166.0 1.75 1.88 1.61 156.3 175 1.35 170 1.90 160 1.90 135 0.90 Uric Acid * 5 190 190 174.0 1.80 1.60 1.57 152.4 180 1.50 170 1.00 140 1.95 Casein 6 215 195 185.0 1.92 1.47 1.56 151.4 190 1.73 185 1.67 165 1.27 160 1.32 Cottonseed Meal .... 6 180 180 160 155 155 135 161.0 1.45 1.25 1.30 1.25 1.10 0.72 1.18 114.5 in the pop corn plants the reverse was true, but the differences in both cases were within the range of error inherent in the method. From these experiments it is clear that in all cases the growth of plants when furnished sodium nitrate was markedly better than when no nitrogen, except that in the seed, was present. The poorest showing for the nitrate was 113.6 per cent of the check in Table XV; the best was 236.6 per cent in Table X. BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 185 Urea The plants grown in cultures containing urea as the source of nitrogen showed in the case of the pop corn a decidedly better development than those in the check solution. This indicates that the nitrogen of urea is available to some extent, but not sufficiently to make urea equal to sod- ium nitrate. However, in the inoculated cultures it proved equal to the nitrate as a source of nitrogen. This indicates that amraonification or some other transformation of urea is necessary for the best utilization and assimilation of that compound by pop corn plants. The weight of the dent corn plants in the sterile cultures showed urea to be about 50 per cent better than the check, though the leaf measurements were no greater J •OE NT C0{ IN- * cm /toc. yres ■ 400 ^-^ / \ / --, ,^00 V / ^ J> ^^ ^^^ ^ ^ ■--^^ ?00 N. ' r^l^ /oo ^^'- C.^-- PW GRAN 5 ?,00 ^z ,-' ■^^ 100 " ■ ^- ^^ — -^^^ <^ ^11^ 000 "S ^=>. " — f K <» 1 c :3 c o -a c r c £ 1 -3i T5 b-2 2 -c o < - in <3 V5 1 <-> -1 < 1 ^ ^ £ 1 __0| is Q >5 si Fig. 2. — A summary of all the dent corn plants grown, showing both the dry weights and the measurements. than those of the check. In the inoculated cultures both length and dry weight showed urea better than the inoculated check, and the dry weight showed even better growth than the dry weight of the plants in the in- oculated cultures of sodium nitrate. A comparison of the sterile and the inoculated cultures, containing urea, shows no difference in the dry weight and the length of leaf is only slightly better in the inoculated cultures. Urea was found unavailable by Pryanishnikov and Lyebyedyev (40), and toxic by Sawa (41). Hovv^ever, it has been reported beneficial by Hutchinson and Miller (16), Molhard (33), Suzuki (52) and Tompson (54). Takeuchi (53) has found that the enzyme, urease, which ammon- ifies urea, is not present in Mays. The com itself then cannot ammonify urea. 186 SOIL SCIENCE Peptone Peptone was utilized by Mays plants in both sterile and inoculated cultures. In the sterile cultures in both varieties of corn it was better than urea but did not quite equal the nitrate as a source of nitrogen. As with urea, the action of B. subtilis seemed to increase its availability for Mays plants. With pop corn, in the inoculated cultures, the growtli was not equal to either that in the nitrate or urea but was considerably bet- ter than in the sterile cultures. With dent corn in sterile cultures the plants make a much better development than in the check but were not equal to those in the nitrate. The growth was 27 per cent better in the inoculated cultures than in the sterile. The plants with peptone were fifth in rank. Hutchinson and Miller (16) have found peptone a source of available nitrogen. Guanin Guanin was found to be exceedingly toxic to the pop corn plants in both sterile cultures and in those cultures which were inoculated with B. subtilis. These plants made practically no growth. In the cultures inoculated with soil water the growth was fair, but as there were only a few plants, no definite conclusions can be drawn. In the sterile cultures it was found to be about equal to the sodium nitrate for dent corn, and was better in the inoculated than in the sterile cultures. The results show clearly that as a source of nitrogen the same chemi- cal compound may have a value differing to a considerable degree for different varieties of a species. Guanin was toxic to pop corn and fur- nished available nitrogen to dent corn. Schreiner and Reed (44) and Schreiner and Skinner (46) have found guanin available to wheat seed- lings. Guanidin Carbonate In the experiments guanidin carbonate was found to be exceedingly toxic to the pop com plants used. Young seedlings made a very slight growth and died within a few days on culture media containing this sub- stance. It was less toxic to dent corn but none of the cultures with this substance were as good as the check. Guanidin carbonate has also been found quite toxic to other plants by Kawakita (18), Schreiner and Reed (44, 45), Schreiner and Skinner (46), and Bierma (4). Benzamid The plants grown in a nutrient solution containing benzamid showed a decidedly toxic effect of this substance. They made a very feeble growth and died within 3 weeks. The action of B. subtilis did not alter the toxicity of this substance. Lutz (30) found that all compounds con- taining the benzin ring group were toxic to plants. BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 1S7 Caffein The caffein nutrient solution with dent corn was more toxic than the guanidin carbonate, and much poorer than the solution used in the check culture. The leaves of the plant were small and pale in color. Those plants grown in the inoculated cultures were slightly better than those in the sterile cultures but the difference was not very marked. This com- pound has also been reported toxic by Lutz (30). Glycocoll GlycocoU was used as the source of nitrogen in only one series of ex- periments. The results of this series showed that it was favorable to the growth of Mays plants. It did not prove equal to sodium nitrate in the single series in which it was used. The growth was very little better in the sterile cultures. Its effect on other plants has been ascertained and found favorable by Schreiner and Skinner (46), Hutchinson and Miller (16j, Dachnowski and Gormley (12), Lefevre (24), Molliard (34), and Schreiner and Reed (47). Uric Acid Thompson (54) has shown by his experiments that uric acid furnishes as good a source of nitrogen for oats as does urea and sodium nitrate. The results of the author's experiments with Mays are very similar. In the sterile cultures the growth of the dent corn was equal to that in the sodium nitrate both in length of leaves and in dry weight. Uric acid was better than urea as a source of nitrogen. There was only 1 cm. difference by measurement and .05 gm. by weight, between tlie averages of the sterile and the inoculated cultures in uric acid. Diphenylamin Diphenylamin was the most toxic substance used. When germinated seedlings were placed upon the agar medium containing this substance the roots turned brown and the plants died within 24 hours. Alanin The results of the experiments with alanin as a source of nitrogen, presented in Tables X, XI, XIV and XV, show it to be a good nitrogen source. In the sterile cultures the plants are nearly as good as those in the corresponding nitrate solution. The plants grown in the inoculated nitrate cultures are better than those of the sterile and better than the in- oculated nitrate cultures. It is, therefore, a good source of nitrogen for Mays, although Schreiner and Skinner (46) founds it slightly toxic to wheat seedlings and Molliard (34) reported it toxic to roots, while Le- fevre (24) found it favorable as a nitrogen source. 188 SOIL SCIENCE Ammonium Sulfate Ammonium sulfate was used in Series V and VII of the experiments. It has been known from the time of Liebig to be very readily assimilated by some plants. In the experiments here reported it was found to give a better growth of Mays than most of the other substances tried, and much better than sodium nitrate or urea. The measurements of the leaves show the sterile cultures to be slightly better while the weights reverse the ratio. Asparagin Asparagin is a substance fotmd very widely distributed in plants, and the results obtained in these investigations show it to be an excellent source of nitrogen for Mays. The plants grown in this solution in sterile cultures are shown by measurements to be surpassed only by those in ammonium sulfate ; by weights they are far better than any others. The growth in inoculated cultures is about equal to that in sterile. It has also been found readily assimilated in the experiments of Baessler (2), Mol- liard (33, 34), Nakamura (37), and Skinner and Beattie (51). Guanidin Nitrate The effect of guanidin nitrate upon Mays v^ras about parallel to that of guanidin carbonate ; approximately the same growth was obtained, the former shovv^ing about the same toxic reaction as the latter. There was a difference in weight of onl}^ .02 gm. between the plants in the sterile cultures inoculated with B. subtilis. Hemoglobin Hemoglobin is a complex animal protein, and it might be expected that, due to the molecular structure, the nitrogen would not be available for plants. The results show that in the sterile cultures it was slightly better than the check both by measurements and weights, but not as good as sodium nitrate. However, in the inoculated cultures the growth was about 25 per cent better than in, the sterile, and much better than the in- oculated check and nitrate cultures. The plants in this culture were among the best of all the cultures. A part of these plants supphed v^ith hemoglobin were grown in very poor light and this may have had some detrimental influence, but even under such conditions they did exception- ally well. Casein Casein, like hemoglobin, is an anim.al protein, and might be thought to be unavailable for plant nutrition. Kelly (20) found that it may be readily ammonified by soil bacteria. The author's experiments show that in the sterile cultures it is favorable, about equal to sodium nitrate, and that in the inoculated cultures it is considerably better than the nitrate as a source of nitrogen. The inoculated cultures made a greater develop- ment than the sterile. B RICH AM— ASSIMILATION OF ORGANIC NITROGEN 189 Linseed Meal That such products as Hnseed meal and cottonseed meal might be used as a source of nitrogen was suggested by Kelly (19). The results here reported show that plants furnished with linseed meal make a slightly bet- ter growth in the sterile cultures than the check plants, but not equal to that of the plants having sodium nitrate. The inoculated cultures with linseed meal were decidedly better than the sterile and also better than the inoculated nitrate cultures. Cottonseed Meal The results w4th cottonseed meal were very similar to those with lin- seed meal. That is, in the sterile cultures the growth was only slightly better than the check but in the inoculated it is markedly better, and the plants here were among the best of all the cultures. Malt The plants grown in the solution to which malt had been added m.ade approximately the same growth as those in the check in both the sterile and the inoculated cultures. This substance furnished practically no nitrogen, nor did the bacteria have any influence on the availability of the inoculated nitrate cultures. Creatin Creatin was used only in Series VII. This compound was of some value as a source of nitrogen, as indicated by the growth, which was somewhat better both in the sterile and in the inoculated cultures than the respective check cultures. There was little difference between them in growth in the sterile and in the inoculated cultures when the creatin was used as the source of nitrogen. Chemical Groups The inorganic nutrient salts, sodium nitrate and ammonium sulfate were both highly beneficial to plant growth as has already been stated, but they were excelled by some of the organic compounds. Among the organic compounds used were three purin derivatives. One, uric acid, was found available and decidedly beneficial, another guanin, was also found favorable to dent corn, while the third, caffein, containing three methyl groups, was quite toxic. The amids of the simple organic com- pounds are shown to contain nitrogen available for plant growth. Gly- cocoll and alanin are amids of acetic and propionic acids, respectively. Asparagin is a moiiamid of amido succinic acid and was one of the most favorable substances experimented with. Urea might be considered a diamido-carbonic acid, the simplest of all the organic acids. The albu- minoid substances peptone, casein and hemoglobin were also available for plant nutrition. 190 SOIL SCIENCE The guanidin derivitives, guEinidin carbonate, guanidin nitrate and creatin appeared to furnish the plant with no nitrogen. The first two were noticeably toxic in their action, while creatin seemed free from toxic properties. Two compounds of the benzin ring group were used, benzamid and diphenylamin, the former with one, the latter with two benzin rings in the molecule. Both of these compounds were highly toxic to the Mays plants. The results with these two compounds are in accord with the work of Lutz (30) who has reported that benzylamin, diphenylamin, analin, and naphthylamin, members of the benzin series, were all toxic to the plants he employed. Of the ground seeds, cottonseed meal and linseed meal contained available nitrogen for the plants, while malt was of no value as a source of nitrogen. The results of this work and that of other investigators lead us to be- lieve that some substances containing organic nitrogen may be used as a source of this element for plants in general. The fact that plants under experiment can absorb some of the substances, without first being broken down, indicates that this can take place with the plants in the fields since they grow in soils containing manure or other decaying vegetable and ani- mal matter. Some of the substances then, in fertilizers, are directly as- similable by the plants and do not need to be ammonified and nitrified as is usually thought. Also, products probably occur in the intermediate stages of decomposition that may be directly utilized by plants. This is contrary to the general belief in agricultural practice that plants must be furnished with either ammonium compounds or nitrates. Nevertheless, most of the substances tried were utilized better or more rapidly when acted upon by B. subtilis. This is intelligible if B. subtilis causes ammoni- fication of such substances, since ammonium sulfate was better than sodium nitrate. Conclusions The results of the investigations reported in this thesis warrant the following conclusions : 1. Zea Mays directly assimilates and uses the following organic nitro- genous compounds named in the order of their availability, asparagin, casein, cottonseed meal, hemoglobin, linseed meal, uric acid, peptone, guanin, alanin, urea, creatin, malt and glycocoll. 2. The following organic nitrogenous compounds are toxic to the growth of Zea Mays: guanidin carbonate, guanidin nitrate, diphenylamin, caffein, and benzamid. Guanin is toxic to pop corn but not to dent corn. 3. Eight organic substances which were directly available produced better growth when acted upon by B. subtilis, probably because of am- monification. These were peptone, guanin, alanin, linseed meal, cotton- BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 191 seed meal, casein, hemoglobin and urea. The last showed this effect only with pop corn. 4. The availability of the following substances was not increased by the action of B. subtilis: urea in the case of the dent corn, sodium nitrate, asparagin, ammonivim sulfate, uric acid, malt, creatin, glycocoll, and those compounds which were toxic. 5. In the case of dent corn 6 substances were better than sodium nitrate ; cottonseed meal, linseed meal, casein, hamoglobin, uric acid, and asparagin. The following, though available, were not better than sodium nitrate: urea, peptone, guanin, alanin, and creatin. 6. The different varieties of the same species of corn react differently with some nutrient substances. Guanin was toxic to pop corn but avail- able to dent corn. Peptone was better utilized by dent corn than by pop corn. 7. The compounds of the benzin ring were found exceedingly toxic to the plants tried. 8. Ammonium sulfate is a far better source of nitrogen for dent corn than sodium nitrate, and is surpassed only by casein and asparagin, when tested by the dry v/eight, and only by asparagin when tested by length of leaves produced. 9. Generally, those organic compounds of high complexity in com- position are better after ammonification, while those of a low degree of complexity are not improved by ammonification. 10. Very likely nitrification following ammonification would be detri- mental, since sodium nitrate was not equal to ammonium sulfate for dent corn. 11. The method of measuring growth by length of leaves gave re- sults very nearly parallel to those obtained by determining the dry weight, and is much simpler. These conclusions apply to the two varieties of com plants used. Only experiments on other species and varieties will show how they react to these substances. Literature Cited (1) Atkinson, Geo. F. 1911. Mushrooms, p. 289-290. New York. (2) Baessler, p. 1884. Assimilation des Asparagins durch die Pflanze. In Landw. Vers. Stat., Bd. 33, p.231~240. Abs. in Hutchinson and Miller (16). (3) Berthelot, M. 1888. Sur la transformation dans le sol, des azotates en composes organi- ques azotes. In Compt, Rend. Acad. Sci. [Paris], t. 106, p. 638. (4) BlERMA, S. 1909. Die Assimilation von Ammon-, Nitrat- und Amidstickstoff durch Mikroorganismen. In Centbl. Bakt. [etc.], Abt. 2, Bd. 23, p. 672-726. 192 SOIL SCIENCE (5) BOUSSINGAULT, JOSEPH. 1835. Recherches chimiques sur la vegetation-Entreprises dans le but d'examiner si les plantes prennent de I'azote a atmosphere. In Compt. Rend. Acad. Sci. [Paris], t. 6, p. 102-112; et t. 7, p. 889-891. (6) BOITSSINGAULT, JOSEPH. 1854. Recherches sur la vegetation-Enterprises dans le but d'examiner si les plantes fixent dans leur organisme I'azote qui est a I'etate gazeux dans I'atmosphere. In Ann. Chim. et Phys., t. 52, ser. 2, p. 5-60. (7) BOUSSINGAULT, JOSEPH. 1855. Recherches sur la vegetation. In Ann. Chim. et Phys., t. 53, ser. 3, p. 149-223. (8) BOUSSINGAULT, JOSEPH. 1856. Recherches sur la vegetation-De Taction du saltpeter sur le develop- pement des plantes. In Ann. Chim. et Phys., t. 54, ser. 1, p. 5-41. (9) BOUSSINGAULT, JOSEPH. 1860-61. Agronomic, Chimie Agricole et Physiologic. Ed. 2. Paris. Ci^ed by Jost (17), p. 133-134. (10) Cameron, Chas. A. 1857. On urea as a direct source of nitrogen to vegetation. In Rpt. Brit. Ass. Adv. Sci., v. 44, p. 44-45. (11) Combes, Raoul. 1912. Sur une methode de culture dest plantes superieures en millieux steriles. In Compt. Rend. Acad. Sci. [Paris], t. 154, p. 891-892. (12) Dachnowskt, a., and Gcrmley, R. 1914. The physiological water requirements and growth of plants in gly- cocoll solutions. In Amer. Jour. Bot., v. 1, p. 174-185. (13) GODDARD, H. N. 1913. Can fungi living in agricultural soil assimilate free nitrogen? In Bot. Gaz., v. 56, p. 249-305. (14.) Harrison, F. C, and Barlow, B. 1907. The nodule organism of the Leguminosae — its isolation, cultivation, identification and commercial application. In Centbl. Bakt. [etc.], Abt. 2, Bd. 19, p. 264-272; 346-441. (15) Hellriegel, H., and Wilfarth, H. 1888. Untersuchungen tiber die Stickstoffnahrung der Graminen und Leguminosen. In Beil. Ztschr. Vereins Riibenzuckerindus.. (Ber- lin). Cited by Jost (17), p. 237. (16) Hutchinson, H. B.,and Miller, N. H. J. 1911. The direct assimilation of inorganic and organic forms of nitrogen by higher plants. In Centbl. Bakt. [etc.], Abt. 2, Bd. 30, p. 513-547. (17) Jost, L. 1907. Lectures on Plant Physiology. Oxford. (18) Kawakita, Y. 1904. Behavior of guanidin to plants. In Bui. Col. Agr., Tokyo Imp. Univ., v. 6, p. 182. Abs. m Hutchinson and Miller (16). (19) Kelley, W. p. 1915. The biochemical decomposition of nitrogenous substances in soils. Hawaii Agr. Exp. Sta. Bui. 39. (20) Kelley, W. P. 1916. Some suggestions on methods on methods for the study of nitrifica- tion. 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Pasteur, t. 4, p. 213-234; 257-275. (57) WiNOGRADSKY, S. 1895. Recherches sur I'assimilation de I'azote libre de I'atmosphere par les microbes. In Arch. Sci. Biol. (St. Petersb.), v. 3, p. 297-352. (58) WiNOGADSKY, S. 1902. Clostridium Pastorianum seine Morphologic und seine Eigenschaf- ten als Butter saureferment. In Centbl. Bakt. [etc.], Abt. 2, Bd. 9, p. 43-54. (59) WiNOGRADSKY, S. 1904. Die Nitrifikation. In Lafar's Handb. Techn. Myk., Bd. 3, p. 132-181. (60) Wolf, W., and Knop, W. 1866. Notiz iiber die Stickstoffhaltigen Nahrungsmittel der Pflanzen. In Chem. Centbl., n. f. Jahrg. 11, p. 774-775. PLATE I Fig. 1. — One of the 1000-c.c. culture flasks used in growing the com plants ; show- the rolled cotton plug and through it passing the glass tube, in which the plant grew through the cotton plug. Fig. 2. — The 168 dent corn plants of Series VII. The flasks to the left of each niunber were sterile and to the right inoculated with B. suhtilis. The various compounds used are : O, distilled water; I, check; II, sodium nitrate; III, urea; IV, peptone; V, guanin; IX, alanin; X, ammonium sulfate; XI, aspara- gin; XIII, uric acid; XVI, hemoglobin; XVII, casein; XVIII, linseed meal; XIX, cottonseed meal; XX, malt; XXI, creatin. Brigham — Assimilation of Organic Nitrogen Plate I W^^- ' ! 1 ■ i ._-r' P m 1 CBM^^hH ■i«*rfj><,' f i 1 1 l^ Soil Science Vol. ITT, No. 2 Brigham — Assimilation of Organic Nitrogen Plate II Fig. 1 Soil Science Fig. 2 Vol. III. No. 2 PLATE II Fig. 1. — Dent corn plants grown under sterile conditions of Series V, with differ-? ent forms of nitrogen added to the stock solution, namely, I, check ; II, sodium nitrate; III, urea; IV, peptone; V, guanin: VI, guanidin carbonate. Fig. 2. — Continuation of figure 1 : IX, alanin ; X, ammonium sulfate ; XI, asparagin ; XII, glycocoU; XIII, uric acid. LIBRARY OF CONGRESS 002 756 470 7