S 593 1.L5 Copy 1 UNIVERSITY OF CALIFORNIA PUBLICATIONS IN AGRICULTURAL SCIENCES Vol. 1, No. 7, pp. 141-172 April 25, 1914 STUDIES ON AMMONIFICATION IN SOILS BY PURE CULTURES BY C. B. LIPMAN AND P. S. BURGESS UNIVERSITY OF CALIFORNIA PRESS BERKELEY UNIVEESITY OF CAIilFOBKIA PUBLICATIONS Koto. — ^Tbe Univeisity of Callfomla Publications are offered in exchange for the pnbU- Mtions of learned gocleties and institutions, universities and libraries. Complete lists of all the publications of the tTnlversity ■will be sent upon request. For sample copies, lists of publications and other information, address the Manager of the University Press, Berkeley, Oalifomla, U. S. A. All matter sent in exchange should be addressed to The Exchange Department, University Library, Berkeley, California, U. S. A. OTTO HABBASSOWITZ B. FBIBDLAEKDEB & SOHN LEXPZIG BFiRTiPT Agent for the series in American Arch- Agent for the series in American Arch- »eology and Ethnology, Classical Philology, aeology and Ethnology, Agricultural Sd- Education, Modem Philology, Philosophy, ences, Botany, Geology, Mathematics, Path- Psychology, ology. Physiology, Zoology, and Memoirs. AGEICULTUBAL SCIENCES. — Charles B. Lipman, Ernest B. Babcock, and John W. Gilmore, Editors. Price per volume, $3.50. VoL 1. 1. The Distribution and Activities of Bacteria in Soils of the Arid Eeglon, by Charles B. Lipman. Pp. 1-21. October, 1912 ?0.ao 2. Studies on the Phenoldisulphonlc Acid Method for Determining Nitrates in Soils, by C. B. Lipman and L. T. Sharp. Pp. 23-37. October, 1912. .16 3. The Effects of Calcium and Magnesium Carbonates on Some Biological Transformations of Nitrogen in Soils, by W. P. Kelley. Pp. 39-49. December, 1912 — - .10 4. The Aluminum Seduction Method as Applied to the Determination of Nitrates in "Alkali" Soils, by Paul S. Burgess. Pp. 51-62. May, 1913 ~ - 15 5. Studies Upon Influences Affecting the Protein Content of Wheat, by G. W. Shaw. Pp. 63-126. October, 1913 ...„ - _ _ .75 6. The Effect of Copper, Zinc, Iron and Lead Salts on Ammonificatlon and Nitrification in Soils, by C. B. Lipman and P. S. Burgess. Pp. 127- 139. March, 1914 15 7. Studies on Ammonificatlon in Soils by Pure Cultures, by C. B. Lipman and P. S. Burgess. Pp. 141-172. April, 1914 25 Vol. 2. 1. Studies in Juglans I. Study of a New Form of Juglans Calif arnica Watson, by Ernest B. Babcock. Pp. 1-46, plates 1-12. December, 1913 60 AGBICULTUBE. — The Publications of the Agricultural Experiment Station consist of Bul- letins and Biennial Eeports, edited by Professor Thomas Forsyth Hunt, Director of the Station. These are sent gratis to citizens of the State of California. For detailed information regarding them address The Agri- cultural Experiment Station, Berkeley, California. BOTANT.— W. A. SetcheU, Editor. Price per volume, $3.50. Volumes I (pp. 418), II (pp. 360), HI (pp. 400), completed. Volumes IV and V In progress. Vol. 1. 1. A Botanical Survey of San Jacinto Mountains, by Harvey Monroe Hall. Pp. 1-140; plates 1-14. June, 1902 »1.00 2. Two new Ascomycetous Fungi Parasitic on Marine Algae, by Minni e Eeed. Pp. 141-164; plates 15-16. November, 1902 26 3. Algae of Northwestern America, by William Albert Setchell and Na- thaniel Lyon Gardner. Pp. 165-418; plates 17-27. March, 1903 2.25 VoL 2. 1. A Eeview of Califomian Polemoniaceae, by Jessie Milliken. Pp. 1- 71; plates 1-11. May, 1904 - .76 2. Contributions to Cytological Techniciue, by W. J. V. Osterhout. Pp. 73-90; 5 text-figures. June, 1904 — .26 3. Limu, by William Albert Setchell. Pp. 91-113. AprU, 1905 _ .26 4. Post-Embryonal Stages of the Laminarlaceae, by William Albert SetcheU. Pp. 115-138; plates 13-14. April, 1905 -. .25 5. Eegeneration among Kelps, by William Albert Setchell. Pp. 139-168; plates 15-17. July, 1905 80 6. A New Genus of Ascomycetous Fungi, by Nathaniel Lyon Gardner. Pp. 169-180; plate 18. July, 1905 „ 15 7. Teratology in the Flowers of some California Willows, by William Warner Mott. Pp. 181-226; plates 16-20. December, 1905 50 8. 0, 10, 11. (In one cover.) The Resistance of Certain Marine Algae to Changes in Osmotic Pressure and Temperature. The R51e of Os- motic Pressure in Marine Plants. On the Importance of Physiolog- ically Balanced Solutions for Plants. The Antitoxin Action of Potassium on Magnesium. By W. J. V. Osterhout. Pp. 227-236. March, 1906 - ^ 12. Cytological Studies in Cyanophyceae, by Nathaniel Lyon Gardner. Pp. 237-296; plates 21-26. November, 1906 _ „ 1.00 V UNIVERSITY OF CALIFORNIA PUBLICATIONS IN AGRICULTURAL SCIENCES Vol. 1, No, 7, pp. 141-172 April 25, 1914 STUDIES ON AMMONIFICATION IN SOILS BY PURE CULTURES BY C. B. LIPMAN AND P. S. BURGESS The study of the physiological efficiency of soil bacteria rather than their number is admittedly the dominant method in soil bacteriological investigations. In view of this fact it is singularly striking to note how little work has been accomplished in the study of some phases of the physiological efficiency of pure cultures of certain groups of soil bacteria. This is especially so, since the introduction of radical changes in our soil bacterio- logical methods have made it necessary to repeat some, if not all, of the work which had been carried out by the old methods. However this may be, it remains a fact that, since the publication of Marehal's^ splendid work on ammonification in solution cultures, but scant information has been adduced from studies of soil bacteria which relate to the physiology as well as the physiological efficiency of even the more common ammonifying bacteria. Certainly, the work carried out along this line in direct soil cultures, which recent work has shown to be so far superior to solution cultures, has been very meager indeed. In order, therefore, to glean some useful information relative to the physiological efficiency of pure cultures of a number of ammonia-producing bacteria, the writers deemed it wise to select a number of organisms and to compare their power to produce ammonia not only from one form of organic matter, but from several forms, most of which have foiind use in farm prac- tice as fertilizers. Accordingly, the following organisms in pure 142 University of California Publications in Agricultural Sciences [Vol. 1 culture were selected for the experiments: B. mesentericus vul- gatus, Ps. putida, B. vulgatus, B. megatherium, B. mycoides, B., suhtilis, B. tumescens, Sarcina lutea, B. profeus vulgaris, B. icteroides, B. ramosus, Streptothrix, sp., Ps. fluorescens, B. vul- garis (Novy strain), Mic. tefragenus. The organic materials experimented with were dried blood (13.16% N), tankage (9.62% N), cotton.seed meal (5.5% N), sheep and goat manure (2.137o N), peptone (14.14% N), fish guano (8.63% N), and bat guano (3.96% N). ilETHOD OP Experiments Fifty-gram portions of soil were placed in tumblers and thoroughly mixed with the organic material to be tested. The tumblers were covered with Petri dish covers and sterilized in the autoclave at a pressure of thirty pounds for three hours. After cooling, the soils were each inoculated with a 1 ec. suspen- sion of the organisms to be tested, made up by shaking with some sterile water a young slope culture grown on bouillon agar. The soil was then stirred with a .sterile spatula after enough sterile water had been added to make a moistui-e content in the soil about equal to the optimum. The soil cultures thus pre- pared were incubated at 28° to 30° C for twelve days. After the incubation period the soils were transferred to copper dis- tilling flasks, 400 cc. of distilled water and an excess of Mg added, and distilled into standard H/10 HCl. The ammonia was then determined in the usual way. No attempt was made to run all the series with tlie different forms or organic matter at the same time, because only the relative powers of the different organisms to produce ammonia were sought. For the same reason amounts of organic matter were chosen in the different series which would least affect the physical conditions obtaining in the cultures rather than amounts employed which would make the total amount of nitrogen added the same in all series. For the reasons above given, therefore, the effects of the various organisms on any given form of organic material will be treated below as a separate series in the ease of each soil and 1914] Lipman-Burgess : Ammonification in Soils hy Pure Cultures 143 comparisons between the different series made only where per- missible. The data, moreover, are presented so that the dupli- cate determinations which were carried out in all cases may be compared. The averages of duplicate determinations, however, are also given as well as the percentages of nitrogen in the organic matter which was transformed to ammonia. Three different California soils were tested with each one of the ammonifiable materials. The soils were a sandy soil from Anaheim, a clay loam from Davis, and a black clay-adobe soil from Berkeley. The mechanical and chemical analyses of these soils are given in Tables I and II which follow. TABLE I Mechanical Analyses of Soils Hyd. value Clay Sandy 5.78 Clay-Loam 19.12 Clay-Adobe 31.93 0.2.5 mm. 14.59 40.93 25.77 0..50 1.04 3.35 3.40 1 2.30 6.60 4.77 2 5.28 7.75 7.49 4 9.62 8.78 6.20 8 11.58 8.10 .87 16 4.87 3.30 2.78 32 15.23 4.15 7.66 64 29.40 TABLE 3.07 II 5.44 Chemical Analysis of Soils Insoluble matter Soluble silica Sandy 73.59 11.17 Clay-Loam 53.55 19.77 Clay-Adobe [ 77.84 K,0 .64 .75 .45 Na,0 .15 .11 .07 CaO 1.39 .82 1.05 MgO .93 1.39 1.21 Un,0, .04 .04 .08 Pe=03 5.10 7.56 4.68 AUO3 3.92 10.04 7.79 PA .12 .13 .23 SO3 .02 .03 .08 Water and organic matter 2.88 5.62 5.72 144 University of California Publications in Agricultural Sciences [Vol. 1 The pure cultures of the organisms employed in these investi- gations were obtained by one of us from the bacteriological laboratories of the University of Illinois. Fresh strains of B. subtilis and B. mycoides were, however, isolated by us from California soils in order to check the stock cultures of the same organisms. The same relative powers of producing ammonia were, however, found to obtain with the fresh as with the old, and different strains thus strengthening the validity of the results below discussed. Series I. Experiments with Dried Blood Sandy Soil Two per cent of finely sifted dried blood was added to the soils in this series, or one gram per fifty grams of soil. In the case of the sandy soil three series in duplicate were tried, and the same relation under different conditions having been found to obtain between the ammonifying powers of the different organ- isms, we give only one duplicate set of the determinations. The incubation period was twelve days throughout at a temperature varying between 27° C and 30° C. The results of the ammoni- fication determinations with all the soils using dried blood throughout are given in Table III. The percentage of nitrogen of the total amount added which is made available is also given in every case. The data in Table III most strikingly indicate the .superiority of Ps. putida, B. vulgaris and Sarcina lutca to all other organisms in their efficiency at the production of ammonia from the nitrogen of dried blood. The next fact of singular interest is that B. mycoides, which has, in the hands of several investigators, shown such marked .superiority over other ammonifying organi.sms in the production of ammonia from organic nitrogen in solutions, manifests a relatively low power to transform the nitrogen of dried blood in soil cultures into ammonia. This, moreover, cannot be considered accidental, since different strains of B. mycoides, as above explained, exhibited that same lack of vigor in three duplicate sets of cultures run at different times and under slightly varying conditions of temiicratnro and period of incuba- 1914] Lipmaii-Burgess : Ammoiiification in Soils by Pure Cultures g K J) w a _w td 00 pel bd td W W 51 W a> >< to <© 00 *o --1 ** CO -^ ^ o cji ci to '-' to 10 I—' COoGOOOSOtfkQO*- to CO 00 'O GO 00 ^^ o' 5 ^ C3S S I-" !0 s r^ w 1— > 00 on to to bo l_l ri- to to 00 to h- ** -1 00 Oi *- >■ O *-0 l-> CO 4^ ~qcoi— lorf^cobilo !_. pi ^ pj ^^ p p ; OS O CO 'o O '^ C?l i 01 00 M en Ci *- p en CO CO I— ' to 4^ pi bo CO '^ CO o CO \o o bi '*> o OOOOOD^-OltOOiOiOOht- a -1 Ol OT M 00 to 'iO Co to OJ 01 rf S 1*^ 00 s 00 to s >4^ CO 00 :o 1 CO "O CO CO CO to O 00 CO 00 1-' OS to O *. T9 " O *" I-" ■ ■rJOOOlOOOSOl— 'GO '-' -I O -. CO ■^^ O (>3 o ■^' CO* ci ■^* O t-- O GO ^ Tt< r-l r-l Csl rl s .2 '^>asg53p,.2 2 fc fq pq" m m ri 02 « W pq 1914] Lipman-Burgess: Ammonification in Soils hy Pure Cultures 151 B. mesentericus. Then follow, not far behind B. proteus vulgaris, B. tmnescens, B. ramosus, and B. vulgaris in the order named, the latter being more than 331/3 per cent short of the efficiency exhibited by B. mesentericus. Nearly all of the other organisms fall more than 50 per cent short of the efficiency of the last- named organism under this set of circumstances, namely, sandy soil with tankage. There are, therefore, but five organisms out of the fifteen tested which can be adjudged distinctly efficient ammonifying organisms under these conditions. B. mycoides again exhibits a low efficiency, and not far different from, though slightly below, that manifested by it in the same soil in the dried- blood series. It must be added here that, with the exception of B. mesen- tericus, the efficient organisms in this part of Series II have also shown more or less marked efficiency in the preceding series. The Clay-Loam Soil Most of the organisms tested in the clay-loam soil seem to have found the latter a more congenial medium for ammonifica- tion with tankage than they did when dried blood was present. In this part of Series II, three of the organisms, namely, B. mesentericus, B. vulgatus, and B. tumescens, were not only the most efficient ammonifiers but also about equal in their ammoni- fying power. It appears, therefore, that so far as tankage is concerned B. mesentericus is an equally efficient ammonifier in the clay-loam and sandy soils. B. tumescens has previously established its pre-eminent position among the fifteen organisms as an ammonifier of dried-blood nitrogen in the adobe soil, but B. vulcjatus enters here for the first time as a markedly efficient ammonifier. Only slightly behind the three organi.sms just discussed in their ammonifying efficiency as regards tankage nitrogen in the clay-loam soil are, in the order named, B. suhtilis, B. mycoides, and B. vulgaris. While the latter has manifested its high efficiency in other series above described, the first two organisnLs named for the first time in the work thus far described show marked ammonifying ability. Only three organisms in this part 152 Uitiversity of California Publications in Agricultural Sciences [Vol. 1 of Series II have shown themselves to he really weak ammonifiers, and they are B. proteus vulgaris, B. icteroides, and Mic. tctra- genus. The first and the third of these, it will be remembered, have given evidence of marked efficiency under other circum- stances, but the second has thus far been throughout an organism of low efficiency. It is striking to note the much greater uni- formity which exists in this portion of Series II in the ammoni- fying powers of four-fifths of the organism.s tested than that which obtains in other parts of this series and of other series. The Clay-Adobe Soil Here again we find the great uniformity in ammonifying power between the larger number of bacteria tested which i.s characteristic of the foregoing section of Series II. On the other hand, the clay-loam soil seems to have been a more congenial medium than the adobe soil for the ammonification of tankage nitrogen, for larger amounts of ammonia are produced in it by the same organisms in the same period of incubation. Sarcina lutea shows the highest efficiency as an ammonitter in this part of the series, but is only slightly more efficient than B. iumcscens. The organism taking third place is B. mijcoides which, indeed, is not far behind the other two. Next in order of importance and still very efficient ammonifiers are B. sitbtiliit and Streptofhri.r, sp. The otlier organism.s are considerably weaker ammonifiers than tliose just mentioned and B. icieroidis again proves to be distinctly the weakest. Both Sarcina lulfa and B. tumescens have, as mentioned above under other circum- stances, plainly evidenced their high efficiency as annuonifiers and B. mycoides and B. suhiilis have taken similar positions with respect to tankage in the clay-loam soil. Sfrppfothrir, sp.. it will be remembered, has also shown a high ammonifying power before in the case of the day-loam soil when dried blood was used. Comparing the three soils in this series with the same ones in the preceding series, it appears quite clear that taking them by and large, ammonifying bacteria manifest a iinuh liighcr efficiency with high-grade tankage than with dried lilood under similar conditions. Likewise also, in Series II a small number of 1914] Lipmaii-Burgess : Ammoniftcatioii in Soils hy Pure Cultures 153 organisms mostly identical with those in Series I seem to mani- fest a distinetl.y high efficiency which the much larger number of the balance do not in most cases even approach. It is worthy of remark, moreover, that B. mycoides attains or rather ap- proaches in portions of Series II its position of prime importance which has thus far so generally been accorded it among ammoni- fying organisms. Series III. Cottonseed IMeal The cottonseed meal used in this series showed on analysis a nitrogen content eqiial to 5.5 per cent. The experiment in this series was otherwise conducted like those of Series I and II, 2 per cent of cottonseed meal being used, or 1 gram per 50 grams of soil. The results are shown in Table III. Again the efficiency of the organisms tested is much greater in the sandy soil than in either the clay-loam or adobe soils. Sandy Soil In this part of Series III the organism of greatest ammonify- ing efficiency is B. tumescens. Those approaching it closely in efficiency are B. vulgatus, Sarcina lutea, and B. mycoides in the order named. B. ramosus takes fifth place and the next three organisms fall more than 33V3 per cent short of producing the amount of ammonia yielded by B. tuiiuscciis. They are B. meseniericus, B. megatherium, and B. proteus vulgaris. The balance of the organisms show only about half the efficiency of B. Uimescens and in one ca.se, B. icteroides, which has in all pre- vious serie.s shown a very low efficiency, only about 25 per cent of the maximum efficiency is manifested. Taken as a whole, the data given in Table III for the Anaheim sandy soil reflects favorably on cottonseed meal as a source of available nitrogen under the conditions named. Again, the greatest efficiency is manifested in this section of Series III by organisms which in all cases have shown high efficiency in other series above reported. As in the case of the heavy soil in the preceding series with tankage, B. mycoides also shows marked efficiency in the light soil when cottonseed meal is used. 154 University of Califoniia Publications in Agricultural Sciences [Vol. 1 5 !S 01 tD o C-. o o ;i; o -+ -t o o Tf t~ CO C-. ;:::: ^ 2 CO to LO s CO t- lO CD C; ^ O I-« r-. C'l Ci 00 CO 00 CD 00 CO CO CD 'CM .-H ,-t (M CO CD □0 CO CO (X t-- CO .-H t- Ol t-- CO (M LO G\l o ^ g ?;! 1.1 s CO a-. CO CO S : : o : ira CO en m uc Tt* CO CO CO ^ --I " =o I ; '^ '-' ^ T-H 1-. (M rl T-H nH CD rH lO O »0 CO T-( C-] Ol T-H t^ rj< (M I- CD CO Ci 00 CO 00 I.- CO i-l ci 00 lO 00 t- o r- CO CO CiC^Cit^OCOf— tlOLOCO ci .-< 00 oi .-i oi -^ o lo 00 2 =s 1 S c I, g ■■§ M c ^ o .3 o< CL, pq « pq pq m 02 pq PQ pq M Ph m S 1914] Lipman-Burgess : Ammonification in Smls by Pure Cultures 155 The Clay-Loam Soil The most notable thing in this part of Series III is as above intimated, the very low efficiency of all of the organisms tested. Indeed only two organisms manifest any notable activity as ammonifiers of nitrogen in cottonseed meal in the clay-loam soil as a medium. These two in the order of their importance are B. ramosiis and Streptothrix, sp. To these, in view of the dis- agreement of the duplicates as above shown, B. vulgaris may probably be added, and perhaps also B. vulgatus. The other organisms are all distinctly below the first two mentioned, and the lowest efficiency thus far noted is that exhibited by Mic. tetragemis in this soil. Considering the high efficiency of the latter organism in Series I, the results just discussed are puzzling. The Clay-Adobe Soil Even a casual glance at the data obtained in the clay-adobe soil as a medium indicates the distinct superiority of that medium to the clay loam soil for ammonification of the nitrogen of cotton- seed meal. Not only relatively but absolutely the data obtained show the production of much larger amounts of ammonia in this portion of the work. As is the case in the sandy soil with cottonseed meal as the ammonifiable material, B. iumescens .shows its distinct superiority to the other organisms as an ammonifier in the adobe soil. The next most efficient organism is B. subtilis, which, however, is con- siderably less efficient ; and the next two organisms, about as far below B. subtilis in efficiency as the latter is below B. iumescens, are B. ramosus and B. mycoides. The other organisms are all low in efficiency, though in nearly all cases absolutely better than the same organisms in the clay-loam soil. Thus far we find that B. subtilis shows itself markedly efficient, for the first time, in the clay-adobe soil with cottonseed meal. All the other organisms above named have manifested marked efficiency in some parts of the foregoing series. Looking at Series III as a whole it is interesting to note that the nitrogen of cottonseed meal seems to be made available through the activity of pure cultures of ammonifying bacteria. 156 University of Calif ornia Publications in Agricultural Sciences [Vol. 1 with much greater rapidity than has heretofore been believed. The next striking fact brought out in these results is the clearly indicated superiority of the clay-adobe soil to the clay-loam soil as a medium for the amnionification of the nitrogen in cottonseed meal. The third point worthy of mention in Series III is the fact that we find again in it. as in the preceding series, only a few of the fifteen organisms tested which show marked ammonifying efficiency. The culture of I's. fluoresce iis died in the midst of these investigations and was not replaced. Series IV. Fish Guano Fish guano, in accordance with the teachings of Voorhees and other agricultural chemists, has always been esteemed a good source of available nitrogen ; in the words of Voorhees," ' ' rank- ing in availability well up to blood and tankage." It seemed to us therefore of importance to compare in these pure culture studies fish guano with the otlier organic materials discussed above. Accordingly a series was started similar to those above described, except that li/i grams of finely sifted fish guano was the ammonifiable material used per 50 grams of soil. The fish guano used contained 8.63 per cent nitrogen. The re-sults obtained are shown in Table VI. The data in Table VI not only seem to confirm the opinion of Voorhees as above stated, if ammonia production by pure cul- tures may be taken as a criterion for determining the availability of fish guano, but they indicate in most striking fashion what was not shown in any of the foregoing series for the fertilizers, and particularly in the sandy soil, namely, the obliteration of the marked physiological diflierences obtaining in other series between the differelit organisms. Of the four nitrogenous fer- tilizers thus far discussed, fish guano seems to contain the form of nitrogen most generally ammonified by a large group of bacteria. 1914] Lipman-Burgess : Ammonification in Soils 61/ Pure Cultures g W 51 cc w td W 50 td W td td td ^ tJ 2.-^ = 00 w 10 t^ 01 CO ^ to 4^ 00 O O) 00 to '^ -^ O l-i to Oi OJ • O I-' CO ►*i h- i-» --1 CO CO Oi OS 01 00 OJ CO -1 -1 h-» O 1-' Oi CS CO ^-J CO o td 2; [H o H 3 M ^ tc OT Ls2 00 CO ro '■0 *^ 10 CO a> i *- 00 00 ta 00 ^ w OJ ^, CO -.1 Ol io CB -1 -^1 CO CO ^ CO *■ s> *^ W OT 00 c ~i 10 to CO CO en CO CO w Ol t-O CO w ^ o '--o ^1 bo i^ 10 CO «:> ! ]58 University of California Publications in Agricultural Sciences [Vol. 1 The Sandy Soil The good ammonifying power manifested by most of the organisms tested in this soil with fish guano finds its maximum in the ease of B. vulgaris, which, it will be remembered, has already given evidence of notable ammonifying efSciency with other nitrogenous fertilizers. B. tumescens, likewise an organism with a record as an ammonifier well established, is a very close second to B. vulgaris in its efSciency at the ammonification of the nitrogen in fish guano. Four other organisms distinctly in the second class, and about alike in ammonifying efficiency in this part of Series IV, are, in the order of their efficiency, Ps. putida, B. vulgatus, B. ramosus, and Sarcina lutea. Peculiarly enoiigh, B. icteroides shows an ammonifying efficiency here which places it in the second class, a position which it has thus far never even remotely approached in the series of experiments above discussed, nor, for that matter, in other series which are described below. All other organisms in this part of Series IV are distinctly- in the third class, but nevertheles manifest notable ammonifying efficiency. In no other series of results have we obtained such .sharp lines of demarcation between the classes of organisms here tested, and arbitrarily grouped, in accordance with their respective powers of transforming organic nitrogen into ammonia. The Clay-Loam Soil The change in the physical condition of the soil from the sand to the clay loam shows a marked effect on the ammonifying power of the same organisms. Nevertheless we find, on the whole, the best set of results thus far obtained with the clay loam when the series with fish guano is studied. In a class by itself under these conditions is B. megatherium, which is markedly superior to all other organisms in this part of Series IV, except B. mescn- tericus, which is a close second. The organism, however, which takes third place, B. vulgaris, falls about 20 per cent short of attaining the efficiency of B. megatherium under these conditions. Distinctly lower in efficiency in this part of Series IV are. in the order of tlieir impnrlaiiiT. but only sliiihtly different from 191-1] Lipmaii-Burgess : Ammoiiificatioii in Soils by Pure Cultures 159 one another, B. vulgatus, Ps. putida, B. ramosus, and B. tumes- cens. All of these, however, fall more than 30 per cent short of attaining the ammonifying efficiency of B. megatherium under the conditions here considered. Two other organisms, B. subtilis and Sarcina lutea, fall 40 per cent short of the eiificiency of B. megatherium, and the others all fall far below even that figure. It is singular, here again, that the physical nature of the soil medium employed should so strikingly and so variously influence the efficiency of the ammonifying bacteria. The two organisms which are distinctly superior in ammonifying ability as regards the nitrogen of fish guano in the clay loam soil were only of moderate efSciency with the same form of nitrogen in the sandy soil. And again, the organism which in the latter soil was paramount in its position retreats in the clay-loam soil to third place. The Clay-Adobe Soil This is the first series of those we have thus far considered, as the data in Table VI shows, in which the clay-adobe soil proves, on the whole, to be inferior to the clay-loam soil. Seven organ- isms out of fourteen here show an extremely low ammonifying efficiency, and two others are by no means efficient organisms. That leaves five organisms in this group which may be considered of importance. Of these, B. vidgaris is the mo.st efficient, but B. tumescens is not far behind it. B. suhtilis belongs to the second class in this group and B. protcus vulgaris and B. mycoides to the third class. It is rather unfortunate that it was not possible in this part of Series IV to obtain better agreement between duplicate determinations. It would not appear to us, however, that the discrepancies in question militate agaio.st the justice of the conclusions above drawn. B. megatherium holds a very good place in the sandy and clay loam soils of Series IV and a fair place in the adobe soil. The same is even more strikingly true of B. vulgaris, and in a minor degree this is also true of B. tumescens. The other organ- isms do not manifest such consistent efficiency under the three widely varying soil conditions. 160 Vniversity of California Publications in Agricultural Sciences [Vol. 1 ^Miscellaneous Series For the purpose of comparing sheep and goat manure as well as phosphatie guano with the other organic materials above described and with peptone, it was deemed of interest to obtain data exemplifying the ammonification in the same soil of all the different materials above used with the three additional ones just mentioned. The sandy soil was chosen for this series and when peptone (Witte) was used, 0.5 gram of it was added to 50 grams of soil. The peptone contained 14.14 per cent N. The sheep and goat manure (2.13 per cent N), owing to its low nitrogen content, was added to the extent of 3 grams per 50 grams of soil, and the phosphatie guano (3.96 per cent N) was also added at the rate of 3 grams per 50 grams of soil. The results obtained are recorded in Table VII. As was to be expected, the very available form of nitrogen in the peptone allows of the production of much larger quantities of ammonia than do the l&ss available forms of the other materials. The fineness of division and easy solubility of the peptone, as well as the form of nitrogen which it contains, doubtless have con- tributed to the results. The more or less uniform decomposition of it, however, by most of the organisms tested shows peptone to be unsuited, as has been claimed by other investigators, for ammoni- fication studies with pure or mixed cultures when the application of the data obtained, to field conditions, is contemplated. How- ever that may be, Sarcina lutea shows the highest efficiency at ammonifying peptone nitrogen and at least six other organisms approach it rather closely. Kelatively speaking, all but two of the organisms tested are efficient ammonifiers of peptone nitrogen. But their position with respect to peptone, as can be seen from Table VII, is no criterion as to their efficiency with respect to the other materials. One of the interesting facts about the ammonification of the sheep and goat manure by pure cultures is that only one organism showed ammonifying efficiency worthy of the name, and that was B. megatherium. The other organisms showed a very slight power only of ammonifying the nitrogen in it. Just why this large discrepancy should exi.st with respect to this manure between B. )ii( (jiilln riiiiii and the otlier orgaiiisiiis still remains 1914] Lipman-Burgess : Ammonification in Soils iy Pure Cultures g tfl ►d CO td M td fo ca _w w _w _W ^ W a tE- § S g S a I 3 g I eg ,| ^ S ^ « -5 3 -t. 2 5" 3 = r t-" to CO p Ol ti) bi ^-' Ci W CO to to CO to to CO to 00 00 to 00 Ci CO CO CO ^1 i-» 00 : cj CO t^ c: t^0 to ^ lOl cn CO C5 CO OS en CO «-> o CO to Ol OS »4^ O 00 O to I-* to tC tO) 00 to CO to to to Ci ►**" *^ Ci Ol 0'( -^ GQ ^ 2 S CO CO CO ■^ Id ^1 O I-- '^' en en CO ■ ai '— ' o <-* to t-O I-* t-0 cc to O wl ^ I-" to tot -a o to --0 > CO CO t-O ■S> I—' to ; 162 University of California Publications in Agricultural Sciences [Vol. 1 to be explained. If, therefore, the readine.ss of the transformation of its nitrogen into ammonia by pure cultures of ammonifiers is to be taken as a criterion, sheep and goat manure must be adjudged to contain a relatively unavailable form of nitrogen. The most amazing evidence portrayed in Table VII is the very high availability of the nitrogen of bat guano, which is a phosphatic guano. While without question the fact of the large amount of the guano used, as well as its low content of nitrogen, preclude an accurate and wholly justifiable comparison of it with the other nitrogenous materials, one cannot help being struck by the large transformation of its nitrogen into ammonia which nearly all of the organisms tested can accomplish. In many ways, the transformation of the nitrogen of bat guano into ammonia resembles that of the transformation of peptone nitrogen. The differences between the ammonifying powers of the different organisms are, however, unquestionably more marked in the case of the bat guano. In its efficiency as an ammonitier of bat guano nitrogen, B. mycoides appears for the first time in all the series studied to be distinctly superior to all the other organisms tested. B. vulgaris easily takes second place. Not far behind, however, and about equal in efficiency, are B. megatherium, B. vulgatus, B. tumescens, and Mic. tetragenus. In the third class are B. ramosus, B. suhtilis, and Ps. putida, B. proteus vulgaris, and B. mesentericus. In the fourth class are Streptothrix, sp., Sarciiia lutea, B. icteroides, and Ps. fluorescens. A Comparison op the Relative Availabilities of the Organic Materials above Employed based on the Percentage op Nitrogen Contained in Them that vv^as Transformed to Ammonia Thus far we have been considering only the relative degrees of efficiency as ammonifiers of the different organisms among them- selves as respecting a "given organic form of nitrogen in a given soil. There is possible, however, a further very interesting study of the data above given as a basis. We refer to the percentage of nitrogen which is transformed in the different materials into ammonia so as to give them a relative rating as to availability as 1914] Lipmait-Burgess : Atninonification in Smls by Pure Cultures g to i-d CO pj pd td vj pj td td W td ^ bd pl s: OS -a 05 ffl i-j o I-. o> h-. OS *- to o " ^ ^ CO h-1 b tl^ CO bi b b b CO b b to b CI 03 *^ CO >(^ o rf» OO *- If- CO p. O ■z CO h-J H w o o b 00 '*- pi ^ 05 *- ji o b S 2 ^ ?r m o w w 03 00 -o *- to g T) 2! "^ h3 lO g 01 s 4^ h^ Ol to oo 03 ^ *• OT c; -■1 bo >f- •^1 o K) (W 'f- if- O) bo jf^ r> w ■p b Ol b CO b £. s p M -^1 CJI to a> i-o CO ~~i Oi ^ o i-t 4^ LO 3 cr >-' 'co O CO w -1 O -1 ■p i-o I-* i-" ^1 OD to ►**> Cl ■rj O -1 >-' I-* Ol -1 Ci CJI tc CS CC •— ' to CO CO l-i -T to CO CI I-* l-i CO w 'I '-" p' H CO •s Q I -1 g s CO p^ o ? 3 *~' " > < --1 OS 4^ *. O i-" I-' ** C^i CO J*^ to *- -O -0 3 Ol -^ 00 CO Ol ro 00 q5 If- III) to OO ^1 £ o o If- ~1 o ^ o o 'to o CI Ol g ^ ^ 10 CO 164 University of California Publications in AqriruUural Sciences [Vol. 1 regards the work of pure eulture.s. The cokimns on availability of the various tables show a comparison in different soils of all the materials as attacked by the same organisms, and give the pereentagas of nitrogen in those materials which were trans- formed to ammonia. In Table VIII all of the columns indicating percentages of nitrogen of different fertilizers made available are brought together from the other tables and one is enabled to compare with much greater ease the different materials on the basis of availability in the same soil and with the same organism and in different soils, with different organisms. TJic Sanrhj Soil So far as this soil is concerned, the data in Table VIII indicate clearly the superiorit.y, from the point of view of the availability of its nitrogen, of cottonseed meal to the other organic nitrogenous fertilizers with which it is compared. Not only are the absolute amounts of ammonia produced in most cases larger from cottonseed meal nitrogen than from other forms, but there are more organisms of the fifteen tested which can vigorously ammonify this form of nitrogen. So that to illustrate, there are but five organisms which have shown the power to transform 15 per cent or more of the nitrogen of dried blood into ammonia in the sandy soil. Under similar conditions there are ten organisms which hold such a record for cottonseed meal. Tank- age .shows itself to be the next important nitrogenous fertilizer to cotton.seed meal from the point of view of the availability of its nitrogen. Thus, comparing it with dried blood as above, we find that there are nine organisms which transform 15 per cent or more of the nitrogen of tankage into ammonia. No such high availability is obtained at all in the case of the fish guano. When we consider these nitrogenous fertilizers from the point of view of the transformation of 10 per cent or more of their nitrogen into ammonia, we find that there are thirteen of the fifteen organisms tested which possess that power as regards cottonseed meal nitrogen and another comes very close to that point. In the ease of tankage nitrogen there are thirteen organ- isms with a similar power. In the case of dried-blood nitrogen 1914] Lipmnn-Burgess : Ammonification in Soils by Piire Cnltures 165 there are but ten organisms which can accomplish that task, and but seven such in the case of fish-guano nitrogen. From the point of view of availability by pure cultures therefore in the sandy soil the four nitrogenous fertilizers are to be rated as follows: cottonseed meal, tankage, dried blood, and fish guano. The first two are nearly alike and are far superior to the last two, which are nearly alike, but much more different from each other than the first two. The greatest efficiency at ammonification manifested In* any organi.sm in the sandy soil is that of B. mesentericus with tankage nitrogen, which transforms 32.52 per cent of the nitrogen present into ammonia in twelve days. It should be noted in this con- nection also that absolutely higher amounts of ammonia are produced from tankage nitrogen than from any other form in the sandy soil, even if there are fewer organisms which attack it readily than there are in the case of the cottonseed meal. Tlie Clay-Loam Soil Conditions are evidently entirely different for ammonification in this soil. Not only is the ammonia production very low so far as all the fertilizers are concerned, but they no longer bear to one another the relation which obtained between them in the sandy soil. There are thus but few organisms which possess the power of transforming 10 per cent or more of the total nitrogen in any of the four fertilizers into ammonia in twelve days. In fact, there are none such in the case of the dried blood, only two such each in the cases of cottonseed meal and fish guano, and seven such in the case of the tankage. No organism attains to the production of ammonia equivalent to 13 per cent of the total amount present in the clay-loam soil regardless of the kind of fertilizer at its disposal. The tankage, however, is superior to the cottonseed meal in the clay loam and distinctly so as above indicated. The cotton- seed meal takes second place, the fish guano third place, and the dried blood is by far the poorest. Indeed, no organism was capable of producing an amount of ammonia in excess of 6.91 166 University of California Publications in Agricultural Sciences [Vol. 1 per cent of the total amount of dried-blood nitrogen furnished, and that occurred in only one ease, all the other organisms pro- ducing much less. The Claij-Adobe Soil Some very striking facts become apparent when the avail- ability of the four fertilizers in clay-adobe soil are considered. AA-Tiile on the whole dried-blood nitrogen is only slightly more efficiently transformed into ammonia than in the clay-loam soil, cottonseed meal and tankage, particularly the former, are more vigorously acted on in the clay-adobe soil by most of the organ- isms. Fish guano, while not markedly so, is none the less superior here again to dried blood. Again comparing the different fertilizers on the ba.sis of the amounts of their nitrogen transformed into ammonia by the organisms tested, we find that four organisms transform 10 per cent or more of the nitrogen in cottonseed meal into ammonia, a like number accomplish similar results in the case of tankage, and none succeeds in that direction in either fish guano or dried blood. While thus cottonseed meal and tankage appear alike, a study of table VIII reveals the superiority of the former in the larger absolute amounts of nitrogen which are transformed there than in the case of the latter. The first striking fact shown in Table VIII is the marked superiority of the sandy soil as a medium for ammonification by pure cultures. Likewise the added fact of its superiority as a medium for most of the organisms tested must be noted in this connection. The second point of great intei-est is the .surprising fact of the superiority of the clay-adobe soil to the clay-loam soil as a medium for ammonification. From its tenacious nature one would suppose the former type to be a much poorer medium for ammonification than the clay loam and yet it is di.stinctly superior to the latter as regards tankage and cottonseed meal. As regards fish guano, it is slightly inferior to the clay loam and again as regards the dried blood about equal to the clay loam or possibly slightly superior. Considering all the data given in Table VIII from all points of view, tankage must be given first place as regards the avail- Ifll4] Lipmaii-Bur(irxs: Jmmonificdlioii in Soils by Pure Cultures 167 ability of its nitrogen, cottonseed meal easily takes second place and, owing to its superiority in the sandy soil, dried blood takes third place. Considering the superiority of the fish guano to the dried blood in the other soils, however, it is probably fairer to adjudge dried blood and fish guano of equal availability from the point of view of the transformation of their nitrogen into ammonia by pure cultures of ammonifying bacteria. General Discussion Several of the facts which have come to light in the investiga- tions above described demand a word of comment with respect to their general significance. First as regards the relative efficiencies at ammonification of the different organisms tested, we find that there is marked variation. Indeed it is difficult to find an organism among the fifteen tested which consistently stands as the best ammonifier regardless of the soil and the ammonifiable material employed. There are, however, one or two organisms which nearly approach .such a description. In other words, it appears that, viewing ammonification of organic nitrogen from the standpoint of pure cultures, every organism will do best with a definite combination of soil and organic matter. To be sure there are some organisms of those tested, even though they be in the minority, which are consistently weak anmiouifiers. B. icteroides and Ps. fluorescens serve to exemplify such. That B. mtjcoides is by no means always the most efficient of ammonifying bacteria as has heretofore been believed is clearly indicated above. On the other hand, it does possess and manifest marked superiority in certain eases. Thus, for example, while showing poor or mediocre ammonifying power in different soils with dried blood, tankage and fish guano, it manifests great vigor in the case of cottonseed meal and succeeds in making the record for the percentage of nitrogen transformed in the case of bat guano, in which it transforms to ammonia 36.06 per cent of the nitrogen present. 168 Vniversity of CaUfortiia Publications in Agricultural Sciences [Vol. 1 Comparing the organisms in any one given soil as a medium we find some interesting facts. In the sandy soil, for exami)lt;, with dried blood B. vulgaris is the most efficient ammonifier. making available or transforming into ammonia 24.36 per cent of the nitrogen present. With tanl^age, B. mesentericus shows the highest efficiency, transforming 32.52 per cent of the nitrogen present into ammonia. "With cottonseed meal, B. tumescens is paramount, yielding an amount of ammonia equivalent to 24.30 per cent of the amount of nitrogen present. With fi.sh guano, B. vulgaris again manifests its superiority over the other organ- isms by changing 13.09 per cent of the nitrogen present into ammonia. When bat guano is used, B. mycoides stands distinctly superior to all others, as above shown, by transforming 36.06 per cent of the nitrogen present into ammonia. In the case of sheep and goat manure there is but one efficient organism and that is B. megatherium, which transforms 24.97 per cent of the nitrogen present into ammonia. Lastly when peptone is used Sarcina lutea stands pre-eminent, and when all materials are compared, regardless of whether they were used in all soils or not, the last- named organism makes the record for availability by transform- ing 41.98 per cent of the nitrogen present into ammonia. There are thus six organisms out of the fifteen which make records in one and the same soil but with different forms of organic matter. One of the six stands superior in the cases of two nitrogenous materials, namely dried blood and fish guano, and that is B. vulgaris. Comparing the same organisms with the same nitrogenous materials, above iised, except the last three named, in the clay- loam soil, we find that with dried blood B. proteus vulgaris is most efficient, transforming 6.9 per cent of the nitrogen present into ammonia. With tankage, B. tum,escens is pre-eminent and transforms 12.74 per cent of the nitrogen present into ammonia. With cottonseed meal, Streptothrix, sp., is superior, transforming 12.84 per cent of the nitrogen present into ammonia ; and lastly with fish guano, H. megatherium is again pre-eminent, trans- forming 11.66 per cent of the nitrogen present into ammonia. We see again, therefore, that in one and the same soil, with foiir different materials, four different organisms make records for 1914] Lipman-Burgess: Am mollification in Soils by Pure Cultures 169 availability. To be sure, two of these organisms have shown themselves superior to all others in the sandy soil but with different materials. In the case of the clay-adobe soil we find that with dried blood B. tumescens stands at the top of the list and transforms 9.41 per cent of the nitrogen present into ammonia. With tank- age in the same soil, Sarcina lutea is the most efficient ammonifier and transforms 12.44 per cent of the nitrogen present into ammonia. With cottonseed meal, B. tumescens is again superior to all others and transforms 18.20 per cent of the nitrogen present into ammonia; and finally with fish guano B. vulgaris again assumes the ascendency and transforms 8.64 per cent of the nitrogen present into ammonia. B. tumescens is very nearly as efficient in this latter case as B. vulgaris. We have thus seen that organisms which in all cases have shown their superiority in other soils are also very efficient in the clay-adobe soil. It is clear, therefore, that only about half of the fifteen organisms tested show greatest efficiency in some soil or with some form of organic matter. Scrutinizing more closely the efficiencies of each of these, we must concede to B. tumescens the paramount place among them, for it has stood pre-eminent, in five combinations of soil and fertilizer, out of fifteen, and has in addition been close to first place in several other instances. Comparing our findings with those of Marchal, the following critical statements must be made. First, that results of solution cultures are no criterion as to results to be obtained in soils. Secondly, that no two forms of organic nitrogen are attacked and ammonified with the same vigor by any one organism. Thirdly, that different soils will modify an organism's power to ammonify any one given form of nitrogen very markedly, so that it may be efficient in one case and feeble in another. Fourthly, that the ammonifying efficiency of organisms is greater in sandy soil, and possibly in others, than in solutions, for we have obtained a transformation of 41.98 per cent of peptone nitrogen and 36.06 per cent of bat guano nitrogen into ammonia by Sarcina lutea and B. mxjcoicles respectively in twelve days at temperatures between 27° C and 30° C, while Marchal only obtained similar transformations in thirty days at 30° C in albumin solutions. 170 University of California Publications in Agricultural Sciences [Vol. 1 Owing to the general distribution of the efficient ammonifying organisms above described in most soils, it is extremely improb- able, to judge from our results, that we may look forward to a profitable form of inoculation of soils with ammonifying bacteria. The choice, however, of the form of nitrogenous fer- tilizer for a .soil, which will be most readily made available, when available nitrogen is needed, may indeed be something of much greater practical significance, as our data would appear to indicate. Particularly emphatic are our results in illustrating that, at least so far as pure cultures are concerned, and as also partly shown by J. G. Lipman^ and others, with mixed cultures, prevalent ideas with respect to the relative availability of common nitrogenous fertilizers are incorrect. Both tankage and cotton- seed meal, and, in some cases, fish guauo, show higher avail- abilities than dried blood, which we have always been in the habit of regarding as the most available of organic nitrogenous fertilizers (based only on vegetation tests). Nor do we argue too far afield from our subject when we make the remarks just preceding. We recognize fully that availability as measured by ammonification does not necessarily denote availability as measured by nitrification nor by assimila- tion of nitrogen by plants. We cannot, however, help noting, also, that a persistent preference exists among practical orchard- ists in this state for tankage as against dried bood and we therefore feel that, judged by other critera, similar conclusions, must be drawn to those adduced from our experiments with pure cultures of ammonifying bacteria. We have decided, however, to go much further in these experi- ments and are now prosecuting more elaborate investigations on nitrogen transformation not only to ammonia but also to nitrates. In these studies we shall deal with the soil flora as existing in a large variety of soil types freshly collected from the field for our purposes. Moreover, we shall employ the raw, unsifted fertilizer material just as it is used by the farmer. From these investiga- tions we hope to glean much more evidence which will be of great practical significance, and, like the results above described, also of marked scientific interest. 1914] Lipman-Btirgess : Ainmonifiration in Soils by Pure Cultures 171 Summary Results ai'e above given which deal with : 1. The marked differences in ammonifying efficiency of fifteen organisms in pure culture. 2. The soil was used as a medium and three types employed, sandy, clay loam and clay adobe. 3. Pour common fertilizers as sources of nitrogen were used, in all soils, and peptone, bat guano, and sheep and goat manure employed only in the sandy soil, besides. The four fertilizers were dried blood, tankage, cottonseed meal and fish guano. 4. The nature of the soil, as well as the nature of the nitro- genous material, markedly modify an organism's ammonifying power. 5. There is no regularity in these variations and they cannot be foretold. 6. While it is difficult to make an exact decision, B. tnmescens appears, on the whole, to have been the most efficient organism above tested. 7. The highest efficiency in a single culture with a fertilizer was shown by B. mrjcoides, which transformed in twelve days at 27° to 30° C, 36.06 per cent of the nitrogen in bat guano into ammonia. 8. The highest efficiency in a single culture with peptone was shown by Sarcina lutea, which transformed 41.98 per cent of the nitrogen present into ammonia under similar conditions. 9. A comparison of availability of nitrogenous fertilizers with ammonifiability as a criterion, according to our experiments, markedly changes the positions of tankage, fish guano, and cottonseed meal with respect to dried blood, showing them in most or in many cases to be superior to the latter. 10. One half of the number of organisms tested is far superior to the other half in ammonifying ability. 11. Interesting comparisons with ilarchal's work are made. 12. B. icteroides has shown itself throughout to possess but little ammonifying efficiency. 172 Vnivcr.siti) of Californiii Publieationa iit Ayricnltural Sriences [Vol. 1 13. Experiments are following those above described to make the latter more complete. Elaborate tests are being made witti many soil types using the original soil 's mixed flora from freshly collected samples in the field. Not only ammonifying powers of these mixed flora in the different soil types will be studied, but also the corresponding nitrifying powers. A large variety of nitrogenous fertilizers as employed in practice will be tested here. REFERENCES OP MORE OR LESS DIRECT PERTINENCE 1 Bull. Acad. Roy. Soc. Belg., 3 ser., vol. 2.5, p. 727. 2 Voorhees, Fertilizers, Maemillan Co., 1898. 3 N. J. Sta. Bulletin, no. 246. 4 Ann. Sci. Agron., vol. 19, p. 209. 5 Rpt. Del. Exjx Sta., 1899, p. 76. 6 Cent, f iir Bakt., 2" Abt., vol. 20, p. 322. ^ Ibid., vol. 15, p. 433. s Ibid., vol. 29, p. 238. Ibid., vol. 31, p. 49. UNIVEESITY OF OALIFOENIA PUBLICATIONS— (Continued) 13. On a Small Collection of Mosses from Alaska, by J. Cardot and T. Th^riot. Pp. 297-308; plates 27-28. December, 1906 10 li. Some Unreported Alaska Spbagna, together with a Summary of the Cryptogamic Work of the University of California Botanical Ex- pedition to Alaska in 1899, by William Albert Setchell. Pp. 309- 315. September, 1907 05 16. On Nutrient and Balanced Solutions, by W. J. V. Osterbout. Pp. 317- 318. October, 1907 „ 05 16. A Synopsis of the North American Godetias, by Willis Linn Jepson. Pp. 319-354; plate 29. December, 1907 40 Indes, pp. 355-360. Vol. 3. 1. Compositae of Southern California, by Harvey Monroe Hall. Pp. 1- 302; plates 1-3, with a map. December, 1907 3.00 2. The Origin, Structure, and Function of the Polar Caps in Smilacina amplexicaulis Nutt., by H. D. Deusmore. Pp. 303-330; plates 4-8. December, 1908 _ 35 3, 4. (In one cover.) The Value of Sodium to Plants by Reason of Its Protective Action. On the Effects of Certain Poisonous Gases on Plants. By W. J. V. Osterhout. Pp. 331-340. June, 1908 „ 10 6. Contributions to the Knowledge of the California Species of Crusta- ceous Corallines, I, by Maurice Barstow Nichols. Pp. 341-348; plate 9. December, 1908 _ _ 10 6. Contributions to the Knowledge of the California Species of Cnista- ceous Corallines. II, by Maurice Barstow Nichols. Pp. 349-370; plates 10-13. April, 1909 „ 15 7. New Chlorophyceae from California, by Nathaniel Lyon Gardner. Pp. 371-375; plate 14. April, 1909 _ 10 8. Plantae Mexicanae Purpusianae, by T. S. Brandegee. Pp. 377-390. May, 1909 _ 16 Index, pp. 397-400. Vol. 4. 1. Studies in Ornamental Trees and Shrubs, by Harvey Monroe Hall. Pp. 1-74; plates 1-11; 15 text-figures. March, 1910 „ .76 2. Gracilariophila, a New Parasite on Gracilaria eonfervoides, by Harriet L. Wilson. Pp. 75-84; plates 12-13. May, 1910 10 3. Plantae Mexicanae Purpusianae, H, by T. S. Brandegee. Pp. 85-95. May, 1910 _ 10 4. Leuvenia, a New Genus of Flagellates, by N. L. Gardner. Pp. 97-106; plate 14. May, 1910 _ 10 5. The Genus Sphaerosoma, by William Albert Setchell. Pp. 107-120; plate 15. May, 1910 _ - 16 6. Variations in Nuclear Extrusion Among the Fucaceae, by Nathaniel Lyon Gardner. Pp. 121-136; plates 16-17. August, 1910 „ .16 7. The Nature of the Carpostomes in the Cystocarp of Ahnfeldtia gigarti- noides, by Ada Sara McFadden, Pp. 137-142; plate 18. February, 1911 05 8. On a Colacodasya from Southern California, by Mabel Effie McFadden. Pp. 143-150; plate 19. February, 1911 06 9. Fructification of Macrocystis, by Edna Juanita Hoffman. Pp. 151-158; plate 20. February, 1911 06 10. Erythrophyllum deles.ierioides J. Ag., by Wilfred Charles Twiss. Pp. 159-176; plates 21-24. March, 1911 16 11. Plantae Mexicanae Purpusianae, m, by T. S. Brandegee. Pp. 177-194. July, 1911 15 12. New and Noteworthy California Plants, I, by Harvey Monroe Hall. Pp. 195-208. March, 1912 16 13. We Hydrophyllaceen der Sierra Nevada, by August Brand. Pp. 209- 227. March, 1912 ^0 14. Algae Novae et Minus Cognitae, I, by William Albert Setchell. Pp. 229-268; plates 25-31. May, 1912 _ 40 15. Plantae Mexicanae Purpusianae, IV, by Townshend Stith Brandegee. Pp. 269-281. June 26, 1912 ~ 15 16. Comparative Development of the Cystocarps of AntitlMmnion and Frionitis, by Lyman L. Daines. Pp. 283-302; plates 32-34. March, 1912 20 17. Fungus Galls of Cystoseira and Ealidrys, by Lulu M. Estee. Pp. 305-316; plate 35. March, 1913 ~ 10 18. New Fucaceae, by Nathaniel L. Gardner. Pp. 317-374; plates 36-53. April, 1913 V 75 19. Plantae Mexicanae Purpusianae, V, by Townshend Stith Brandegee. Pp. 375-388. June, 1913 15 Index, pp. 389-397. LIBRftRY OF CONGRESS iin ivil lliilllllHllllll': tmiVEBSITY OF OALIFOEinA PUBUOATIONB— 002 781 525 P C Vol. 5. 1. Studies in Nicotiana I, by William A. Setcbell. Vp. 1-86; plates 1-28. December, 1912 „ 1.26 2. 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