UNIVERSITY OF CALIFORNIA PUBLICATIONS 
 
 IN 
 
 AGRICULTURAL SCIENCES 
 
 Vol. 1, No. 1, pp. 1-20 Issued October 15, 1912 
 
 THE DISTRIBUTION AND ACTIVITIES OP 
 
 BACTERIA IN SOILS OF THE 
 
 ARID REGIONS^ 
 
 BY 
 
 CHARLES B. LIPMAN 
 
 INTRODUCTION 
 
 The student of soils in the humid region, when for the first 
 time exploring soils in the arid region, is invariably struck with 
 the extraordinary depth of the latter as against the very shallow 
 nature of the former. Taken by and large, and excepting the 
 faulty soils, including those underlaid at no great depth by stiff 
 clay, coarse gravel, hardpan, or original rock, respective^, the 
 soils of the arid region very commonly show a depth of at least 
 eight to ten feet, and, when viewed in section, exhibit such a 
 striking uniform it.y in texture and color as to attach to this 
 unusual condition, in the mind of the observer, a certain marked 
 practical and scientific interest. The full significance to crops of 
 the arid region of this extraordinary condition in our soils was 
 first realized and pointed out by Hilgard and was made the 
 subject, by him and Loughridge, of a comprehensive investiga- 
 tion on the ''soil-columns" of California, a large part of which 
 is completed, but some of which is still in progress. The study 
 of the soil-columns of California comprised what might be 
 looked upon as a very thorough partial soil survey of Cali- 
 fornia. It was the intention of the investigators above named, 
 at the inception of the work, to obtain columns of soil repre- 
 senting depths of twelve feet, including a sample for every foot 
 
 * Eead before the Society of American Bacteriologists, Washington, 
 D. C, December 27, 1911. 
 
2 University of California Publications in Agrindtural Sciences \ Vol. 1 
 
 ill depth, and to obtain a kno\vled<iT of the chemical constitu- 
 tion and the texture of the soils by makino^ systematic chemical 
 and mechanical analyses of all the samples thus collected. The 
 information thus obtained in the several years in which the soil- 
 columns Avere studied by Ililj^ard and Louo'hrid^e and the large 
 number of types of soils considered, alons: Avith the most striking' 
 circumstance of the depths to which plant root-systems of the 
 arid regions penetrated, led Hilgard to believe that the striking- 
 chemical and mechanical differences between the soils of the arid 
 and humid regions, as well as the differences in the development 
 of the root-systems in these regions, respectively, might find 
 a parallel also in a difference between the bacterial flora at 
 various depths in the soil. It was this belief on Hilgard's part 
 and his valuation thereof as being of exceeding scientific interest 
 as well as practical value, that led to the association with him 
 something over three years ago of the writer, and it was then 
 that I undertook, among other biological problems in soils, a 
 study of the nitrogen-transforming and nitrogen-fixing bacteria 
 in the different layers of soils in the arid region. This study. 
 while it has progressed considerably, is still in the first stage 
 of its development and the complete results thereof are intended 
 ultimately to be combined with the mechanical and chemical 
 analyses of these soils in a comprehensive report on the whole 
 work. For the purposes of this paper, it is sufficient to give a 
 resume of some of the important results obtained in these inves- 
 tigations with an account of the methods employed in the work, 
 so that it may serve as a preliminary communication on the sub- 
 ject and bring out certain striking facts with reference to the 
 distribution of bacteria in California soils. 
 
 :\rp7rTT0T)s employp]d in these investigations 
 
 One of the most difficult problems in connection with these 
 investigations was to find a method for the collection of soil 
 sam[)les at the several depths which would fairly represent the 
 actual conditions which obtain there, so far as the bacterial 
 Hora are concerned. Our first attempts in this direction were 
 made with an auger of the typc^ manufactured by Iwan Brothers 
 at South Bend, Indiana, by means of which we tried, through 
 
1912] Lipman: Bacteria in Soils of Arid Eegions 3 
 
 successive sterilization of the auger (before taking each sample), 
 to obtain a sample which represented, uncontaminated, each of 
 the soils as they are found in their natural state in the field. 
 With a sterile spatula there were taken from the samples thus 
 obtained with the auger representative samples which were im- 
 mediately placed in sterile cotton-stoppered bottles. It was 
 soon found, however, that this method could not be relied upon 
 for accurate results, since no matter how carefully the samples 
 were thus taken, there were many chances for contaminating 
 samples from the lower layers with soil from the upper layers 
 and thus obtaining results which were erroneous. After much 
 experimenting we finally decided on the following plan for 
 taking the soil samples, which, so far as I know, is as free from 
 chances of error as any method that can be adopted in a series 
 of investigations which must of necessity be so extensive. In- 
 deed, I believe the chances for error here are so small that they 
 cannot affect the validity, to any appreciable extent, of the 
 results obtained. Our method consists in having dug, a day 
 or two prior to sampling, a hole twelve feet in depth with at least 
 one vertical wall and large enough for a man to stand in. The 
 samples are taken as follows: With a sterilized spade, a layer 
 of soil of about five or six inches in depth is sliced down along 
 the whole length of the wall which is to be sampled. After this 
 is done, to remove the soil that in the one or two days' exposure 
 may have become contaminated, the fresh surface thus obtained 
 on the vertical wall is sterilized by means of a plumber's torch 
 on the surface surrounding every spot, previously marked off, 
 at which a sample from each foot in depth is to be taken. When 
 this is done a sterile cylindrical tin tube, a little over one inch 
 in diameter and about ten inches long, is driven at right angles 
 to the wall into the spot selected for sampling, immediately 
 drawn out when sufficient soil has thus been obtained, and the 
 cotton plug replaced. In our first experiments, glass tubes of 
 the size described were employed, with paraffined corks at one 
 end and cotton stoppers at the other. We found this to be a 
 poor method, however, and have replaced the glass by tin tubes, 
 closed at one end and plugged with cotton at the other. These 
 are sterilized at 150 deg-rees centierrade for one hour and a half 
 
4 University of California Publications in Agricultural Sciences [Vol. 1 
 
 before using. In this way by the use of a plumber's torch at 
 every depth as we descend from the surface of the soil down 
 to the twelve-foot depth, we obtain, by starting at sterile sur- 
 faces, a sample of soil representing as nearly as possible the true 
 condition which obtains at very depth. The samples are marked 
 properly, taken to the laboratory, and examined for their am- 
 monifying, nitrifying and nitrogen-fixing powers by means of 
 a modified Remy method, the solutions employed for the work 
 being prepared in accordance with the formulae used by J. G. 
 Lipman.^ Every 50 c.c. portion of the medium in a 250 c.c. 
 Erlenmeyer flask is inoculated with 5 grams of soil. 
 
 DESCRIPTION OF SOILS EMPLOYED IN THESE 
 EXPERIMENTS 
 
 The descriptions given below represent the soils which were 
 employed for bacteriological examinations and sampled for the 
 purpose as above described. The numbers employed below are 
 used throughout all the following tables so as to make unneces- 
 sary any further descriptions. 
 
 Soil No. 1. Eed clay loam mesa soil, from Riverside, California, on 
 which good orange trees were growing at time of sampling. The soil is 
 well supplied with potash, but rather poor in phosphoric acid and very 
 poor in humus and nitrogen. It is underlaid by hardpan at six feet from 
 the surface, which continues on down to the twelve-foot depth, "With the 
 careful cultivation which is given it, along with proper fertilization and 
 tillage, the soil produces profitable crops of oranges and lemons. 
 
 Soil No. 2. Silty alluvial loam, from Davis, California. The samples 
 used were obtained from between some fig trees at the University Farm. 
 This soil is practically uniform in color from the first foot to the twelfth 
 and only becomes slightly different in texture below the fifth foot, becoming 
 gradually coarser and sandier as we descend to the lower layers. It is 
 well supplied with potash, phosphoric acid, and lime and has, for a soil of 
 the arid region, a normal content of humus. 
 
 Soil No. 3. Sandy alluvial loam, from Davis, California. Samples were 
 taken from a wheat field at the University Farm, only to a depth of ten 
 feet. This soil is well supplied with j)hosphoric acid, potash and lime, 
 but rather poor in humus and nitrogen. The sand is of a coarse nature 
 and becomes rapidly coarser, descending from the first foot down to the 
 twelfth, where it is found as very coarse sand. 
 
 1 Bulletin 180. N. J. Agr. Expt. Station. 
 
1912] Lipmau: Bacteria in Soils of Arid Eegions 5 
 
 Soil No. 4. Sandy alluvial loam, from Davis, California. Samples ob- 
 tained in almond orchard at the University Farm. This soil is not nearly 
 so coarse as soil No. 3 and shows a more uniform texture throughout a 
 seven-foot depth, but after that becomes coarser in texture. It is better 
 supplied with humus and nitrogen than soil No. 3 and is well supplied 
 with potash, phosphoric acid, and lime. 
 
 Soil No. 5. Alluvial loam, from Davis, California. Samples obtained 
 in a pear orchard at the University Farm. The soil is uniformly of a 
 fine sandy loam texture for a depth of nearly five feet and then rapidly 
 becomes much coarser than the soil at similar depths in No. 3. The upper 
 soil is well supplied with potash, phosphoric acid, and lime, and fairly well 
 supplied with humus and nitrogen. The lower layers are rather poor in 
 phosphoric acid, humus and nitrogen. 
 
 Soil No. 6. Fine silty soil, from Hanford, California. Samples taken 
 from a vineyard at Hanford, from the first to the ninth foot only. No 
 sampling was done below the ninth foot because of the fact that the water- 
 table was reached at about that point and it was almost impossible to get 
 samples uneontaminated. This soil is almost devoid of humus and contains 
 but little nitrogen, but is fairly well supplied with phosphoric acid, potash, 
 and lime. No alkali is present in the soil. 
 
 Soil No, 7. Silty alluvial loam, from Davis, California. Samples taken 
 at the University Farm, close to a young eucalyptus tree, about twenty 
 months old. The soil is fairly uniform in texture throughout the entire 
 depth studied and is fairly well supplied Avith humus and nitrogen, and 
 well supplied with phosphoric acid, potash, and lime. No alkali is present. 
 
 Soil No. 8. Alkali soil from Tulare, California. Taken only to a depth 
 of ten feet, owing to water conditions such as those described in soil No. 6. 
 This soil contains very little humus and is strongly impregnated with salts, 
 especially ' ' black alkali. " It is otherwise well supplied with phosphoric 
 acid, potash, lime, and the other minerals. Hardly any vegetation can exist 
 on this soil after the salts have risen to the upper layers. 
 
 Soil No. 9. A very stiff and tenacious silty clay adobe, from Imperial, 
 California. Uniform in texture from the surface down to the eighth foot, 
 at which there is found a layer of fine sand for a foot and a half in 
 depth and then a silty sand below to the twelve-foot depth. The soil 
 throughout is almost devoid of humus and contains but very little nitrogen; 
 It is very rich, however, in phosphoric acid, potash, and lime. The upper 
 layers of the soil consist of particles of silt and clay which are so fine 
 as to become cemented together into an extremely hard, refractory material, 
 which is almost of the consistency of a dry, but not heated, brick. A 
 considerable quantity of common salt is present in this soil. This soil has 
 never been cultivated or cropped. 
 
 Soil No. 10. Fine, sandy soil from the desert of Coachella Valley. In 
 this, as in the Imperial Valley soils, there are to be found narrow layers 
 of an inch or two, and sometimes more, of very fine shells of former life 
 which existed in the water at one time covering this land. The soil has very 
 
6 University of California Publications in Agricultural Sciences [Vol. 1 
 
 little or no humus and nitrogen. It is, however, rich in phosphoric acid and 
 lime and -well supplied with potash. The soil is uniform in texture through- 
 out the twelve-foot depth and becomes only a little coarser at twelve feet. 
 The only changes visible in color and texture in the vertical wall are 
 merely those of the shell layers above noted. The soil from which these 
 samples w^ere taken has never been cropped, but similar soil, with a good 
 water supply, produces very fine alfalfa. Very little alkali is present in 
 this soil. 
 
 Soil No. 11. Fertile, alluvial loam from Hayw^ard, California. Uniform 
 in texture for seven feet and then rapidly becoming quite coarse and re- 
 maining so down to a depth of twelve feet. This soil is very fertile, 
 producing good crops of cherries, walnuts, potatoes, and other agricultural 
 plants. It is well supplied with humus and nitrogen, judged by the 
 standard for soils of the arid region, throughout the twelve-foot depth. 
 The phosphoric acid, potash, and lime are also plentiful in all the soil layers. 
 The samples used in these investigations were obtained in a cherry orchard. 
 
 AMMONIFICATION IN SOIL COLUMNS 
 
 Second only to the importance of soil bacteria in maintaining 
 the total nitrogen supply in soils is their power to supply con- 
 stantly available nitrogen to plants. The essential nature of 
 this important phase of the activities of soil organisms is in no 
 wise detracted from by the recent research which has made it 
 clear that some plants at least can take their nitrogen from the 
 soil in forms other than the nitrate. While many of them may 
 not absorb their nitrogen in the form of nitrates, it seems quite 
 certain that practically all of them must take their nitrogen in 
 forms much simpler than the proteid. This being undeniably 
 the case, some agency in the soil is necessary to accomplish the 
 transformation of the organic nitrogen (no matter what the 
 source of the latter to the soil may be) into a simpler, more 
 available, or more assimilable form. These agencies we have 
 found to be the various types of soil organisms which constitute 
 what we now designate by the term "ammonifying flora" of the 
 soil. 
 
 With these statements admitted, it seems reasonable to sup- 
 pose that any increase in the activities of the organisms, included 
 under this head, is a distinct advantag^e to the plant. Under our 
 climatic conditions, where, as above stated, the plant roots very 
 deeply, besides making a large lateral root-development, it is 
 necessary to have the activities of the ammonifying organisms 
 
1912] Lipman: Bacteria in Soils of Arid Regions 7 
 
 not only in the upper layers of the soil, but in the lower layers 
 where an actual examination of the root-systems of plants shows 
 a large development of fibrous or feeding roots. A study, there- 
 fore, of the ammonifying powers of the different layers of soil, 
 or, rather, of the microorganic flora which they contain, is of 
 practical moment, since it is bound to throw light on the soluble 
 nitrogen supply for roots in the greater depths of soil and 
 indicate what practical measures may be taken toward sustain- 
 ing and encouraging the growth and activities of the organisms 
 responsible for that soluble nitrogen supply. Since, therefore, 
 we assume ammonia production to be the first great step recog- 
 nized by our analytical methods in the transformation of soil 
 nitrogen, I have first determined the ammonifying powers at 
 various depths of soils, which may be considered typical of well- 
 defined areas and conditions in the arid region. 
 
 For this purpose there were inoculated into sterile 50 c.c. por- 
 tions of 1 per cent peptone solution, 5 grams of soil from every 
 foot from the surface down to the last depth taken, as above 
 described. After four days incubation at about 28 degrees 
 centigrade, the cultures were washed into copper distilling flasks, 
 sufficient distilled water added, as well as a slight excess of 
 magnesia, and distilled. The distillate was caught in standard 
 tenth normal hydrochloric acid, the excess of which was titrated 
 with standard tenth normal ammonia. Table I gives the results 
 of determinations of the ammonifying power of the soils chosen, 
 as above described. The ammonifying power of only one soil, 
 namely No. 6, is not given, for the reason that the soil column 
 had inadvertently become contaminated before we were ready 
 to use it. 
 
 The numbers of the soils refer to corresponding numbers 
 under the descriptions given above, and the amounts of ammonia 
 produced, as given in the table, represent milligrams of nitrogen 
 as ammonia. 
 
 The data given in table I prove very clearly two facts. First, 
 that in the typical deep and normal soils of the arid region, the 
 activities and the distribution of the ammonifying flora seem to 
 run parallel with the texture, the chemical composition, and the 
 root-development in these soils. Second, that in the absence of 
 
University of California Publications in Agricultural Sciences [Vol. 1 
 
 
 
 
 
 
 TABLE 
 
 I 
 
 
 
 
 
 
 
 
 Ammonification in 
 
 Soil Columns 
 
 
 
 
 Soil No, 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 
 mg. 
 
 mg. 
 
 mg. 
 
 mg. 
 
 mg. 
 
 mg. 
 
 mg. 
 
 mg. 
 
 mg. 
 
 mg. 
 
 1st ft. 
 
 59.80 
 
 67.76 
 
 68.25 
 
 61.60 
 
 54.67 
 
 72.05 
 
 7.28 
 
 25.46 
 
 12.15 
 
 42.84 
 
 2nd ft. 
 
 55.46 
 
 77.00 
 
 72.66 
 
 54.46 
 
 49.14 
 
 68.90 
 
 4.90 
 
 8.27 
 
 6.46 
 
 36.84 
 
 3rd ft. 
 
 52.30 
 
 69.02 
 
 68.40 
 
 38.50 
 
 32.76 
 
 70.43 
 
 5.46 
 
 8.20 
 
 3.21 
 
 14.14 
 
 4th ft. 
 
 55.83 
 
 70.63 
 
 43.05 
 
 44.80 
 
 lost 
 
 65.72 
 
 2.80 
 
 8.69 
 
 1,99 
 
 7.28 
 
 5th ft. 
 
 49.72 
 
 67.48 
 
 34.23 
 
 39.90 
 
 8.40 
 
 63.69 
 
 4.76 
 
 4.87 
 
 1,25 
 
 7.84 
 
 6th ft. 
 
 49.00 
 
 84.63 
 
 31.64 
 
 47.60 
 
 15.68 
 
 60.50 
 
 4.41 
 
 8.27 
 
 1,61 
 
 21.19 
 
 7th ft. 
 
 31.70 
 
 72.17 
 
 42.00 
 
 44.80 
 
 17.85 
 
 56.90 
 
 2.45 
 
 6.03 
 
 1,57 
 
 7.14 
 
 8th ft. 
 
 30.54 
 
 52.57 
 
 35.00 
 
 22.54 
 
 9.80 
 
 50.43 
 
 10.22 
 
 5.40 
 
 1.34 
 
 7.48 
 
 9th ft. 
 
 28.65 
 
 52.43 
 
 35.70 
 
 32.90 
 
 15.40 
 
 45.69 
 
 10.64 
 
 2.18 
 
 1,24 
 
 5,60 
 
 10th ft. 
 
 25.40 
 
 36.89 
 
 
 29.40 
 
 10.22 
 
 43.25 
 
 
 2.23 
 
 2,11 
 
 16,38 
 
 11th ft. 
 
 15.65 
 
 21.56 
 
 
 25.48 
 
 11.06 
 
 40.76 
 
 
 1.34 
 
 4.87 
 
 16,64 
 
 12th ft. 
 
 14.30 
 
 35.84 
 
 
 38.22 
 
 10.50 
 
 38.45 
 
 
 2.62 
 
 1.44 
 
 10,85 
 
 humus and moisture, or in the presence of alkali salts, the activi- 
 ties of ammonifying organisms are seriously handicapped. 
 
 To discuss these more in detail we find, for example, in soil 
 No, 1, derived from the mesa soil at Arlington Heights, River- 
 side, a strong ammonification, varying but little from the first 
 foot down to the seventh, below which depth we find a sudden 
 marked decrease in ammonia production, for the reason, doubt- 
 less, that from the sixth foot down to the twelfth we find a 
 layer of hardpan which, owing to its poor aeration and poor 
 water conditions, is unfit for the development of a vigorous bac- 
 terial flora. In other words, we find in this soil-column, through 
 the ammonifying power of the various depths of soil, an expres- 
 sion of the vigor and numbers of bacteria present in these soil 
 layers and also of the amounts of soluble nitrogen which can 
 there be expected to be made available through the agency of 
 soil organisms. 
 
 In soil No, 2, however, which represents a good, deep alluvial 
 soil, we find a very vigorous ammonification from the first seven 
 feet, and only slightly reduced ammonia production in the eighth 
 and ninth feet, after which we find a large reduction of about 
 50 per cent in ammonia production for the other three feet. 
 We have here, therefore, good vigorous ammonia production down 
 
1912] Lipman: Bacteria in Soils of Arid Regions 9 
 
 to the tenth foot and therefore an indication that in these soils 
 there is constantly being made available nitrogen, if organic 
 nitrogen be present from humus and other sources, for the needs 
 of plants with deep root systems. 
 
 In soil No. 3, which is more sandy than the other alluvial 
 soil described and which rapidly becomes coarser in texture as 
 we descend into the lower layers, we find vigorous ammonifica- 
 tion to obtain down to the fourth foot, below which we find a 
 considerable decrease in ammonifying power, owing to the fact 
 that in that coarse soil neither water nor humus, nor soluble 
 minerals, are present in sufficient quantity to encourage bacterial 
 development. Here, however, we find the general tendency for 
 ammonifying organisms to penetrate to the greater depth quite 
 plainly visible. The remarks made for soils 2 and 3 are just 
 as truly applicable to the other alluvial soils from the same 
 district represented by Nos. 4, 5, and 7. The marked produc- 
 tion of ammonia, even in the twelfth foot of No. 7, is in accord 
 with the fine physical and chemical condition of that soil to 
 that depth and therefore deserves additional mention here. 
 
 As to other types of soils, the data in the table show plainly 
 enough what a profound effect strong alkali salts (both black 
 and white alkali salts among them) may exert on the ammoni- 
 fying flora and their vigor. Here ammonification is indeed very 
 feeble in the surface soil, becoming feebler as we go down until 
 the eighth foot is reached, at which depth, as well as in the ninth 
 foot, we find quite a marked increase in ammonia production. 
 This is doubtless due to the fact that the total salt-content is 
 at that depth much lower and therefore not so seriously affecting 
 the activities of the organisms there contained. As for the 
 desert soils, which never have contained much humus and very 
 frequently contain too much alkali, it is natural to expect a 
 rather feeble ammonifying power on the part of the soils. Table 
 I shows that in this case the expected happens. In soil No. 9. 
 for example, not only the lack of humus and moisture, but the 
 very unfavorable physical condition, above referred to under 
 the description of that soil, along with its salt-content, have so 
 far affected the ammonifying power of that soil as to reduce it 
 to a little over one-third of what the normal vallev soils de- 
 
10 University of California Publications in Agricultural Sciences [Vol. 1 
 
 scribed have exhibited. Moreover, it would seem that the salt- 
 content in the lower layers of this soil, which increases as we 
 go down, has very seriously checked the development of these 
 organisms there and was probably assisted by the unfavorable 
 physical condition mentioned. In soil No. 10, while the salt- 
 content is only meager, we have a rather coarse, sandy soil with 
 hardly any humus, which is therefore for that reason an unfavor- 
 able medium for the development of bacteria, to say nothing of 
 the lack of moisture there and the great heat which these desert 
 soils must absorb from the sun. We therefore have a very much 
 smaller ammonifying power in the upper layers of the soil than 
 exists even in soil No. 9, from Imperial, and then a very rapid 
 decrease to almost no ammonifying power in the lower layers. 
 
 By a general survey of all of these data, it would certainly 
 seem that we are justified in drawing the conclusion that am- 
 monification and the ammonifying flora of soils are vigorous for 
 several feet down in the arid region and are limited in their 
 activities only by the presence of large amounts of salt or a 
 lack of humus and moisture. Since, however, California soils, 
 taken by and large, are deep, we have reason, from the facts 
 above given, to suppose that the ammonifying power in most 
 of these soils, which are not in any way "abnormal," is vigorous 
 at great depths. 
 
 NITRIFYING POWERS OF SOIL COLUMNS 
 
 By very many and perhaps by most plants, nitrate is the 
 form of nitrogen taken up. It is therefore of importance not 
 only to study ammonia formation in soils, but nitrate formation 
 as well. In these investigations we have studied qualitatively 
 and quantitativel}^ the production of nitrites and nitrates in 
 ammonium sulfate solution by soils from the different depths 
 in every case. Here also, as in the ammonification work, 5 grams 
 «of soil were used to inoculate 50 c.c. of culture solution. The 
 results obtained in this work are set forth in a qualitative man- 
 ner, as to nitrate formation merely, in table II, since it is 
 sufficient for the purpose of this preliminary paper to know to 
 what depths in the soil nitrates are produced. Later publica- 
 tions, giving the more complete data of these investigations, will 
 
1912] Lipman : Bacteria in Soils of Arid Begions 11 
 
 give the quantitative results as they are given for ammonification 
 in table I. The plus sign represents nitrate formation and the 
 minus sign the absence thereof. The numbers of the soils are 
 referred again to the descriptions above given. 
 
 . TABLE II 
 
 Nitrification in Soil Columns 
 
 Soil No. 1 2 3 4 5 6 7 8 9 10 11 
 
 1st ft. + + + + + + + ___ + 
 
 2nd ft. + + + + + — + — — — + 
 
 3rd ft. + + + + + _ + ___ + 
 
 4th ft. + + + + + _ + ___ + 
 
 5th ft. + + + + + _____ + 
 
 6th ft. trace + — — + — ___ — + 
 
 7th ft. — — — — — — — — — — J^ 
 
 8th ft. _ — _______ — + 
 
 10th ft. — — — — — ___ 
 11th ft. — — — — — ___ 
 12th ft. — — _— _ ___ 
 
 From the data in table II we see again a striking resemblance 
 between nitrification in the soil depths and ammonification in 
 the same. All the alluvial soils in particular show very uniform 
 nitrate formation. The latter seems to be as much inhibited 
 in soil No. 1 by the hardpan layer as is ammonification. In 
 soils 2, 3, 4, and 5, as well as 7, we find a general tendency for 
 nitrates to be formed in the first five feet and then an enfeebled 
 powTr of nitrate formation, in some cases for one foot and in 
 other cases a total loss of that power. In nearly all cases these 
 run parallel with a similar decrease in ammonia formation, but 
 it seems that nitrate formation is more seriously hampered by 
 the conditions which curtail ammonia formation and particularly, 
 it appears, by the lack of oxygen in the lower layers of the soil. 
 This is an account, therefore, of the first case which has come to 
 my notice of nitrification at any depths below two or three feet 
 in the soil, and shows a marked difference in itself between soils 
 formed and existing under humid and those formed and exist- 
 ino: under arid conditions. Nitrogen therefore is available in 
 
12 University of California Publications in Agricultural Sciences [Vol. 1 
 
 these soils, not only for those plants which are able to absorb 
 ammonia nitrogen, but also for that larger class of normal 
 plants which absorb their nitrogen in the nitrate form. It must 
 be said here, however, that nitrate formation proceeded always 
 more rapidly in soils from the first foot than in cultures pre- 
 pared from the other depths. This may indicate a smaller num- 
 ber of nitrifying organisms in the lower layers of the soil or 
 perhaps a less vigorous flora, but their activities are uniform 
 from the second foot down to the last depth in which they 
 show no activity as indicated in table II. In soil No. 6, which 
 w^e find on analysis contained merely a trace of humus, that cir- 
 cumstance seems to have made the soil unfit for the develop- 
 ment of the vigorous bacterial flora and is supported by the 
 data in tables II and III. As to the nitrate formation in 
 culture solutions by the inoculation with this Hanford soil, 
 nitrates were produced only after a month's incubation and only 
 in small quantities in the culture prepared from the upper foot 
 of soil, whereas all other surface soils, Avhen inoculated into solu- 
 tions with the exception of those which show no nitrification at 
 all, showed nitrate formation before the end of two weeks. In 
 culture solutions from soil No. 6, kept about three months and 
 prepared from the lower layers of soil, no nitrates were ever to 
 be found. The depressing effect of alkali on the bacterial flora, 
 as well as the inhibiting effect of a lack of humus, moisture, 
 and the proper physical condition, are again exemplified in 
 table II in soils 8, 9, and 10, as they w^ere for the same soils in 
 table I referring to ammonification. Even after one month's 
 incubation, not one of these soil-samples showed any nitrate 
 formation, whether the culture was prepared with the soil from 
 the upper layers or from the lower. There seems to be a total 
 absence of nitrifying bacteria of one kind or another. 
 
 The best example of the penetration of nitrifying bacteria 
 to great depths was obtained in soil No. 11, a fine alluvial loam 
 from Hayward, where nitrate formation was obtained down to 
 the ninth foot in the soil. In this case also there was, besides 
 a mere formation of nitrates, as shown by a qualitative test, an 
 actually vigorous nitrate formation in the lower layers as well 
 as in the upper layers of the soil. It would seem again here 
 
1912] Lipman : Bacteria in Soils of Arid Regions 13 
 
 therefore, in general, that where soils in the arid region are 
 supplied with a moderate amount of humus, with the proper tex- 
 ture and chemical constitution, as well as freedom from alkali, 
 all of which is true of the large majority of our soils, nitrification 
 as well as ammonification is found to obtain vigorously in the 
 lower layers of the soil for four feet at least, and in some cases 
 to six and to nine foot depths. 
 
 NITROGEN FIXATION IN SOIL COLUMNS 
 
 The next point of interest to determine in these soil-column 
 investigations from the bacteriological standpoint was to show 
 whether or not the supply of nitrogen, at the disposal of the 
 ammonia-forming and nitrate-forming organisms, which we have 
 found developed to such great depths, and enabling roots to 
 have a soluble nitrogen supply there, was provided merely by 
 the humus content of the soil at those depths and produced from 
 decaying roots, or carried dow^n from the upper layers; or, was 
 that nitrogen supply in part a new one obtained directly from 
 the atmosphere by nitrogen-fixing bacteria. If such were the 
 case, we should, of course, have enormous quantities of nitrogen 
 fixed per acre, since the fixation would not be limited to the 
 upper foot of soil. Accordingly, experiments were inaugurated 
 to obtain the facts which exist with reference to this matter. 
 
 Here the necessary mannite solution w^as inoculated with five 
 grams of soil in each case, and a culture prepared from every 
 foot in depth in the case of every soil. Table III shows in 
 tabular form the results obtained, which are set forth qualita- 
 tively. The numbers at the heads of the columns refer again 
 to the numbers used in the description of soils, and one plus 
 sign is intended to show the presence of Azotobacter, two of 
 a fairly vigorous development of these organisms, and three of 
 a very vigorous development. In this qualitative way, there- 
 fore, nitrogen fixation has been judged by the development of 
 Azotobacter as a criterion. It may justly be argued against this 
 that other organisms are capable of fixing nitrogen and that the 
 quantitative figures would be preferable to the qualitative one 
 showing merely the presence of Azotobacter. While this argu- 
 ment may in part be true, it appears from my results, which 
 
14 University of California Fuhlications in Agricultural Sciences [Vol. 1 
 
 show quantitative as well as qualitative figures in these as well 
 as other experiments, that in the absence of Azotobacter only 
 very slight fixations of nitrogen or none are obtained. 
 
 From the results set forth in table III, it appears that only 
 one soil of the eleven tested shows the presence of Azotobacter 
 as deep down as the fourth foot and six others show the pres- 
 ence of these organisms in the third foot. Most of them, how- 
 ever, show the presence of Azotobacter in vigorous form only 
 in the first two feet. It is therefore not sufficient, evidently, for 
 
 TABLE III 
 
 Nitrogen Fixation in Soil Columns 
 
 Soil No. 1 2 3 4 5 6 7 8 9 10 11 
 
 1st ft. +H-+ + + + + + + + + + +++ - + + + — — — + + 
 
 2ndft. ++ + + + + + + + + + + + + — + + + — — — + + 
 
 3rd ft. + — ++ + + + + + + — ++ — — — + + 
 
 4th ft. — — — — + + + — — — — — + 
 
 5th ft. — — — — — — — _ _ _ _ 
 
 6th ft. — — — — — — — -. _ _ _ 
 
 7th ft. — — — — — — — _ _ _ _ 
 
 8th ft. — — — — — — — _ _ _ _ 
 
 9th ft. — — — — — — — _ _ _ _ 
 
 10th ft. — — — — — — — — — — — 
 
 11th ft. — — — — — — — — — — — 
 
 12th ft. — — — — — — — ____ 
 
 soils to be chemically and physically as favorably constituted as 
 these soils are for ammonification and nitrification to encourage 
 the deeper penetration of Azotobacter. As is well known, these 
 organisms are extremely sensitive to a lack of oxygen and it 
 would appear that this circumstance regulates and controls the 
 penetration of Azotobacter organisms as above portrayed. I think 
 in addition, however, it may fairly be argued that the presence 
 of Azotobacter in more than half of these soils in the third foot 
 is, in itself, a favorable indication of the nature of the soils in 
 question. It is of interest also that, in soil No. 5, Azotobacter 
 organisms, with the nitrogen-fixing power as vigorous as those 
 above, were found in the fourth foot. This, so far as the writer is 
 
1912] Lipman: Bacteria in Soils of Arid Eegions 15 
 
 aware, constitutes the only published case of even this extent of 
 penetration of Azofobacter organisms. It has been reported to 
 me, however, that Azobacter organisms have been found in the 
 twelfth foot of soil in some of the very favorably constituted loess 
 soils of Nebraska. The question put in the introduction to this 
 subject of nitrogen fixation is therefore answered in the negative. 
 For the greater depths, at any rate, in which ammonification 
 manifestly is vigorous in our soils, Azotobacter organisms do 
 not penetrate and are not the source of the supply of nitrogen 
 which can be transformed at those depths by the ammonifying 
 organisms or by the nitrifying organisms. The nitrogen supply 
 of these, therefore, in the lower layers of the soil must be the 
 humus produced from the decaying roots at those depths, or 
 the humus brought down in solution from the upper layers of 
 the soil. 
 
 As regards soil No. 6 we have here again a total absence 
 of Azotobacter organisms, possibly due in part, at least, if not 
 wholly, to the absence of any but very small amounts of humus, 
 by which I have already tried to explain the feeble nitrification 
 only in the first foot of this same soil. The same remarks 
 also which were made above, with reference to soil Nos. 8, 9, and 
 10, as regards their ammonifying and particularly their nitrify- 
 ing power, apply again in the case of their nitrogen-fixing power. 
 No Azotobacter organisms and no fixation of nitrogen were ever 
 observed in any of these soils, no matter from what depth of soil 
 the cultures were prepared. 
 
 In justice to this subject it must further be stated here that 
 the comparatively slight penetration of Azotobacter organisms 
 in our soils may be due to factors other than merely a lack of 
 a plentiful supply of oxygen. There is evidently some other 
 circumstance which controls the presence or absence in many 
 of our soils of Azotobacter organisms and that may also limit 
 the depth to which these organisms may penetrate. Just what 
 this factor may be is not at present clear to the writer, but the 
 fact remains that frequently soils with a good chemical and 
 physical constitution and producing good crops, will yet show 
 no Azotobacter organisms. 
 
16 University of California Puhlications in Agricultural Sciences [Vol. 1 
 
 GENERAL DISCUSSION 
 
 As I have already pointed out in an earlier publication,^ the 
 slow formation of clay substances in soils of the arid region, 
 owing to the peculiar climatic conditions there obtaining, is 
 doubtless responsible for a much greater degree of aeration in 
 soils because of the larger volume of pore spaces made possible 
 through a lack of large quantities of cementing substances. Thus 
 when soils first begin to form from disintegrating rock we have 
 much more complete aeration with an encouragement for bac- 
 teria, probably the earliest inhabitants of the soil, to penetrate 
 to greater depths. Such penetration on the part of bacteria is 
 invariably accompanied by the production of more favorable 
 physical and chemical conditions in the soil for the roots of 
 plants. These in their turn, through physical and chemical 
 changes which they bring about in the soil in their search for 
 water and food, make better conditions for a deeper penetration 
 of bacteria and so through mutual aid the latter and the higher 
 plants are able, under our arid climatic conditions, to make the 
 deeper layers of soil a more congenial medium for each other. 
 The changes thus brought about result in a more uniform tex- 
 ture of soils at great depths, uniformity of chemical composi- 
 tion, including humus content, in all the soil layers, and a 
 much closer approximation of the bacterial flora in the lower soil 
 layers to those of the upper layers than can be found in the 
 average soils of the humid region, where climatic conditions are 
 unfavorable to good aeration, because tendencies opposite to those 
 above described for our soils are in operation. An estimate of 
 the biological condition of our deep soils was thus similarly made 
 by Hilgard on a priori considerations and the investigations 
 above recorded serve, in general, to confirm his surmise. 
 
 Viewing the subject in its entirety, we find that the organ- 
 isms forming ammonia in soils penetrate to greater depths than 
 the nitrifying or nitrogen-fixing bacteria studied. While am- 
 monification is usually most vigorous in the surface, four to six 
 feet, it is none the less very pronounced in the lower layers from 
 six to ten feet in depth in all of our normal deep soils. Hardpan, 
 
 - Ijii)rnan, C. B., New Facts about Bacteria of California Soils, Science 
 N. S., June 11, 1909. 
 
1912] Lipman : Bacteria in Soils of Arid Regions 17 
 
 alkali, and a lack of humus and moisture decrease the ammoni- 
 fying powers of our soils or are not favorable to the develop- 
 ment of vigorous ammonifying flora, but their effects are just as 
 pronounced in the upper layers of these abnormal soils as in 
 the lower layers which, therefore, cannot be fairly compared 
 with our deep average soils as to bacterial content. To what a 
 serious extent alkali salts may affect ammonification has been 
 shown by me in a recent paper.^ That the humus content alone 
 may profoundly affect the number and vigor of bacteria is well 
 exemplified in both soils No. 6 and 10, where all other condi- 
 tions but the humus content are favorable and where both the 
 number and physiological efficiency of the organisms is small. 
 
 It would therefore seem, in brief, that ammonification is 
 vigorously active in the lower soil layers in soils of the arid region 
 where humus is present and hardpan and alkali are absent. 
 Since these conditions are complied with in the average of our 
 cropped soils, the opinion is justified that the deep penetration 
 of bacteria is a distinctive characteristic of soils in arid regions 
 which results from much better aeration, as a starting point, 
 than can be attained in soils of the humid region. The experi- 
 mental data above given amply confirm this opinion and help 
 to explain why deep plowing is not only harmless in our soils 
 but directly beneficial, and why three or four feet of upper soil 
 may be removed in grading, and alfalfa and fruit trees may be 
 grown on the newly uncovered subsoil without difficulty, a feat 
 which cannot be accomplished on soils of the humid regions. 
 
 As for nitrification my data present again features of striking 
 interest. They go to prove that nitrate formation, like ammoni- 
 fication, goes on at much greater depths in soils of arid than in 
 soils of the humid region, and thus render distinctly sectional the 
 observations of Dyer* on this subject, and makes them applicable 
 only to soils of the humid region. While the nitrifying organ- 
 isms are doubtless more susceptible to a lack of oxygen than the 
 ammonifying bacteria, the differences obtained above between 
 the two groups of organisms, so far as soil fertility is concerned, 
 are rather those of degree than of kind. The same relationships 
 
 4 Bulletin 106, p. 55, O. E. S., U. S. D. A. 
 3 Centrallblatt fiir Bakt., 2 Abt., vol. 32, p. 58. 
 
18 University of California Publications in Agricultural Sciences [Vol. 1 
 
 displayed by the ammonifying bacteria toward hardpan, alkali, 
 and a lack of humus and moisture, hold in a more exaggerated 
 way as regards the nitrifying organisms. More specifically, the 
 writer has also shown^ the distinct effects of each of the alkali 
 salts on nitrifying bacteria in work quite recently completed. It 
 would appear in general, however, that in our deep soils, a supply 
 of nitrate as well as of ammonia is at the disposal of plants for 
 a depth of five or six feet. As regards the nitrogen-fixing powers 
 of soils of the arid region, my results show plainly that they do 
 not differ strikingly from those of soils in the humid regions, 
 if the presence and vigor of Azotobacter organisms be taken as 
 a criterion. While it is true that in one or two cases Azotobacter 
 organisms were found in our soil-columns below the depth at 
 which they occur elsewhere, and perhaps at a slightly greater 
 depth in all soils in which they were found, I feel loath to believe 
 that these are expressions of a rule for soils of the arid region. 
 Other observations indeed lead me to believe that Azotobacter 
 development has not gone so far in our soils as it has in soils 
 of other regions. For example, I have studied many soils in Cali- 
 fornia with a favorable physical and chemical constitution which 
 were absolutely devoid of Azotobacter organisms. If therefore 
 the results set forth above, with reference to nitrogen fixation, 
 are to be considered representative, the nitrogen supply in the 
 lower layers of the soil must be replenished in this region as 
 well as. in the humid region, not from direct fixation by Azoto- 
 bacter, but from the nitrogen of the upper soil layers. 
 
 With reference to these investigations in general, one or two 
 additional points need more than passing consideration. First, 
 as to the method of collecting the soil-samples for examination, 
 it appears to the writer that every possible precaution was used 
 to prevent contamination and it would be difficult to devise a 
 method which takes into consideration and avoids more of the 
 avenues of contamination by which any results might be vitiated. 
 Moreover, I find strong confirmation of this belief in the facts 
 brought out in the data above given, viz., that any abnormality 
 in the soil was sure to be reflected in the results obtained with 
 cultures prepared from that abnormal soil. Thus hardpan layers 
 
 5 Cent, f iir Bakt., 2 Abt., vol. 33, p. 305. 
 
1912] TApman : Bacteria in Soils of Arid lief/ionn 19 
 
 never ^^ave evidence of vigorous bacteria, nor did alkali soils 
 or soils devoid of humus. 
 
 Secondly, the writer desires to anticipate criticism on the 
 method used in culturin^i' the organisms of the various soil 
 samples, viz., a modified Remy sohition method. No one is more 
 ready than I am to admit the just criticism made of the solu- 
 tion-culture methods in soil bacteriology. Indeed I believe that 
 I was one of the first to put into practice on a lar^ic scale the 
 direct soil-culture method in the laboratory. But when problems 
 of the nature involved in these investigations must be attacked, 
 regard must be had for the chances of contamination in the 
 method employed, and for the feasibility of obtaining, uncon- 
 taminated, large volumes of soil for use in these experiments. 
 When these were considered from all points of view, only one 
 feasible and reliable method of culturing the soils seemed avail- 
 able and that was the solution method. The difficulties, prac- 
 tically insurmountable, which must arise with any other method, 
 when such work is carried out on a large scale as it must of 
 necessity be, can be fully appreciated by those who have ever 
 attempted it. The gratifying results obtained in this work, how- 
 ever, seem to me a further justification of the methods em- 
 ployed. 
 
 It seems of particular moment now, to call the attention of 
 soil bacteriologists in particular, and soil scientists in general, 
 to the important field explored in these investigations and the 
 striking results obtained therefrom, not only because it repre- 
 sents a new field of research, but because it emphasizes more 
 strongly than ever the radical differences which obtain between 
 soils of the humid and arid regions. It also helps to explain 
 the extraordinary appearance of our subsoils (if subsoils they be) 
 and the marvellous root developments of which plants under our 
 climatic conditions are capable. While these studies have not yet 
 departed from the realms of the preliminary, they are replete 
 with facts which are already of considerable practical and scien- 
 tific significance and which are doubtless destined to become 
 more so as time progresses. As a part especially of a comprehen- 
 sive soil study they are invested with unusual importance and 
 may help to solve problems now perplexing and difficult to study. 
 
20 University of California Publications in Agricultural Sciences [Vol. 1 
 
 CONCLUSIONS 
 
 Investigations of the distribution and activities of bacteria in 
 soils of the arid region show: 
 
 1. That samples of soil for studying the flora of each layer 
 of soil can best be obtained from a hole twelve feet in depth 
 with at least one vertical wall, the latter when sterilized being 
 sampled. 
 
 2. That tin tubes ten inches long and about one inch in 
 diameter closed at one end and cotton-stoppered are best for 
 collecting the samples. 
 
 3. That the solution method for stud3ang the soils, despite 
 its many drawbacks, is the most feasible one to emplo3^ 
 
 4. That soils of the arid region at all depths studied show 
 ammonifying powers which, however, are generally most 
 vigorous in the first six or eight feet. In one case ammonifica- 
 tion was noted in soil from a depth of fifteen feet, or adjoining 
 the water-table. 
 
 5. That nitrification is found commonly down to a depth of 
 five to six feet in soils of the arid region. In one case soil from 
 the eight-foot depth showed a vigorous nitrifying power. 
 
 6. That nitrogen fixation through Azotobatcer does not go 
 on below two feet in the soil usually, but has been found in 
 some soils at three feet and in one soil down to four feet. Many 
 soils in the arid region, otherwise favorably constituted, do not 
 contain Azotobacter organisms. 
 
 7. That from the point of view of ammonification and nitri- 
 fication soils in the arid region differ markedly from those in 
 the humid region when the lower layers of soil are considered. 
 The difference is not marked as regards nitrogen fixation. 
 
 8. The results above recorded help to explain the favorable 
 physical and chemical constitution of our soil and also the deep 
 rooting of plants so characteristic of the arid regions. 
 
 Transmitted April S, WIS.