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Reprinted from Sor Scrence, Vol. IV, No. 6, December, 1917
A CORRELATION BETWEEN BACTERIAL ACTIVITY AND LIME REQUIREMENT OF SOILS
FIRMAN E. BEAR Department of Agricultural Chemistry and Soils, Ohio State University
Received for publication September 12, 1917
INTRODUCTION
Limestone regions are noted for their fertility. Alfalfa, red clover, blue- grass, and corn are among the crops which thrive best on limestone soils. Those soils which do not naturally contain carbonate of lime are usually made more productive by applications of lime or limestone. Extensive investiga- tions carried out by the Rhode Island, Maryland, Pennsylvania, Ohio, Tlli- nois, and other agricultural experiment stations have demonstrated the value of lime in either the oxide, hydrate or carbonate form on soils which are acid to litmus. An excellent review of the most important investigational work on the use of lime on acid soils is given by Frear (9).
The investigations of Wheeler at the Rhode Island Agricultural Experi- ment Station, indicate, however, that a number of plants of economic im- portance thrive on soils which contain no solid carbonate of lime. Some of these plants are benefited by lime, but others are injured by applications of lime. Wheeler (36) says that orchard grass (Dactylis glomerata, L.) and mea- dow fescue (Festuca elatior, L.) are less injured by soil acidity than Kentucky blue-grass (Poa pratensis, L.) and timothy (Phleum pratense, L.) and that awnless brome grass (Bromus inermus, L), red top (Agrostis alba var. vulgaris, Thurb.), and Rhode Island bent (A grostis canina, L.) do not seem to be suscep- tible to injury even on decidedly acid soils. He also states (37) that Concord grapes are apparently indifferent to the lack of lime and that cranberries, raspberries, and lima beans are injured by liming, the last named growing splendidly on soils so acid as to entirely destroy lettuce, spinach, onions, beets and asparagus. In his latest publication on this subject Wheeler (38) gives a summary of his work in which he shows that plants vary in their require- ments from those which are injured by applications of lime even to a very acid soil, to those which are unable to live on an acid soil and are benefited remarkably by lime.
Coville (6) states that the blueberry, cranberry, strawberry, blackberry, red respberry, potato, sweet potato, rye, oats, millet, buckwheat, red top, carrot, turnip, cowpea, hairy vetch, crimson clover, soybean, lupine, and serradella are adapted to acid soils. He concludes, “soil acidity is not always
433
SOIL SCIENCE, VOL. Iv, No. 6
434 FIRMAN E. BEAR
an objectionable condition which invariably requires lime” and “under cer- tain conditions, a complete system of acid agriculture is practicable.”
Harter (14) writes that liming has been shown to be beneficial to all crops on Norfolk soils with the exception of beans, peas, and tomatoes. Kossovitch and Althausen (26) report that, while the liming of acid podzol soils strikingly increases the yields, the limit of increase is at about the point of neutralization and that an excess injures the plants. No statement is made as to how the point of neutralization was determined. Heinrich (15) concludes that the determination of lime in a soil, by digesting with 10 per cent hydrochloric acid, can be used as an index in determining what crops will thrive. Ac- cording to his report, the least amounts of lime which will permit of successful growth are:
Calcium carbonate ~*
Crops in the soil ' per cent PPA DInes Oka OCS ALG TY E\ 1G x0 a)-(+ a Js sin els mnie ae aoieseinirinieiaye etsln 0.05 Matsiandupanleyinery emer ratase cies. sniciehs eloeie ets ats scoters apscetete« 0.05 to 0.10 Reads andavetchice: csicvt tomer eraen cite caters lneke So rimieiera cine seleye enaiste 0.10 PREM CLO VEL PEE RETR Ree er Le nA Ree o rene lente eva latieday ohegete Mares 0.10 to 0.12 PA Real facane menor Soy eee ers ea Pl Negar hia "a. Cibialenitech en ae micreye © sabe jolatiele! aleve 0.20 to 0.30
Fred and Graul (10) experimenting with alfalfa, soybeans, and red clover on acid soils of two series, conclude that half enough lime to neutralize the soil acidity as measured by the Truog (32) method is sufficient for the pro- duction of good yields of these crops on acid soils of these two series.
THE RELATION BETWEEN BACTERIAL ACTIVITY AND THE REACTION OF SOILS
One of the reasons usually given for the maintenance of a neutral or slightly alkaline reaction in soils is that the soil microérganisms, which have to do with the processes of decay and the changes by which certain organic and inorganic substances become available for higher plants are unable to work to best advantage in an acid medium. The ammonifying, nitrifying, and nitrogen- fixing bacteria are thought to prefer a neutral or slightly alkaline medium. However, it is probably true that the various groups of soil bacteria are differ- ently affected by the soil reaction. The influence of acidity and alkalinity on the development of pathogenic bacteria has been studied by a number of investigators. The literature on this subject is reviewed quite fully by Itano (21). The degree of acidity or alkalinity which the organisms are able to withstand varies with the species. Certain forms, e.g., Bacterium tuberculosis, are able to live in the presence of a considerable degree of acidity. It is reasonable to believe that soil microorganisms show similar differences in this respect. The fact that many acid soils are supporting vegetation, indicates that bacterial processes are being carried on in them, although these processes might be materially hastened if lime were applied.
The number of bacterial colonies from soil aliquots which will develop on
BACTERIAL ACTIVITY AND LIME REQUIREMENT 435
agar plates is influenced by the reaction of the medium. Hoffmann (16) finds in counting the number of bacteria in soils that a medium slightly acid to phenolphthalein is more favorable than a medium which is neutral or slightly alkaline to phenolphthalein. Fischer (8), who conducted probably the most extensive investigations on the effect of lime on the number of bac- teria in soils, shows that an application of either calcium oxide or calcium carbonate has a very marked effect in increasing the total number of bacteria.
That the rate of ammonification is increased by applications of lime is shown by Voorhees and Lipman (35). Coville (6) points out that many soils acid to litmus contain large amounts of ammonia. Kopeloff (25) shows that ‘““where the soil reaction is unfavorable for the activities of the soil bacteria concerned in ammonification, the soil fungi may prove to be an important compensating factor.”
The rate of nitrification is increased by applications of lime on soils which give an acid reaction with litmus. The results obtained by Lyon and Bizzell (27) are typical. A number of other investigators report similar effects from the use of lime. Scales (29), studying the activities of nitrifying organisms, finds they are most active in the presence of 50 per cent of the calcium-car- bonate requirement (Veitch) of the soil. An excess of calcium carbonate seems to be toxic to the nitrifying organisms. Temple (31) finds that if an organic source of nitrogen is used instead of ammonium sulfate, the formation of nitrates is much greater in acid soils. He explains this increased nitri- fication on the basis of the formation of neutral zones, caused by the production of ammonia, at which points conditions are favorable for nitrification. Temple also shows that calcium salts of organic acids can be used as effectively as calcium carbonate in overcoming the toxic effect of ammonium sulfate on an acid soil. Miller (28), working with a sandy soil acid to litmus, finds that an application of 0.1 per cent of calcium oxide caused a decrease in the ability of the soil to nitrify ammonium sulfate and that 0.5 per cent of calcium oxide stopped the process entirely. Hutchinson (19) finds that calcium oxide acts not alone as a neutralizing agent, but also as a partial sterilizing agent. Since in the experimental work following applications of neutralizing agents are confined to calcium carbonate, it does not seem necessary to include any further discussion on the effect of calcium oxide on the bacterial processes in the soil.
It should be remembered that it has been shown that nitrate nitrogen is not necessary for all plants. Hall and Miller (12) call attention to the fact that ammonium sulfate, on the Park plats of the Rothamsted Farm, pro- duces very good crops of grass, although the soil is deficient in lime and very little nitrification takes place. Hutchinson and Miller (20) find that peas are able to utilize ammonia nitrogen as well as nitrate nitrogen, although the opposite is true with wheat. Kelley (24) shows that rice, grown in swamp land, secures its nitrogen in the form of ammonia. If ammonification proc- esses are less affected than nitrification processes by a deficiency of lime in
436 FIRMAN E. BEAR
the soil, then plants which are able to utilize ammonia can survive where those depending on nitrate nitrogen cannot live.
Hopkins (18) notes that the application of lime increases the power of Bacillus radicicola in certain legumes to fix atmospheric nitrogen. Whiting (39) writes that nodules are often found in abundance on legumes on very acid soils. Japanese clover (Lespedeza) has often been observed by the writer growing on soils strongly acid to litmus and the roots were well supplied with nodules. These nodules were mostly near the surface of the soil. Keller- man and Robinson (22) find that crimson clover inoculation is little affected by the reaction of the soil. Fred and Graul (10) find that, if acid Colby silt loam soil is previously inoculated with B. radicicola, nitrogen fixation by soy- beans is little influenced by applications of calcium carbonate. They also find this true on acid Colby silt loam with red clover. Both clover and alfalfa were able to fix considerable amounts of nitrogen when growing on Colby silt loam and Plainfield sand having only one-half of their acidity (Truog method) neutralized. The Colby silt loam required 10,400 and the Plain- field sand 5200 pounds of calcium carbonate to neutralize one-half ofthe acidity in 2,000,000 pounds of soil. Determinations of the lime requirement (Veitch) on the Colby silt loam soil, chosen from the same locality the year previous, showed a need of 3234 pounds of calcium carbonate per 2,000,000 pounds of soil. The authors state that “the Truog method shows much larger amounts of soil acidity than the Veitch.”’
Ashby (1) shows that the use of lime on the Rothamsted soils more than doubled the nitrogen-fixing power of the Azotobacter. Hoffman and Hammer (17) find that calcium carbonate is essential to non-symbiotic nitrogen fixa- tion, but that the amount required is very minute and was present in sufficient amount in all the soils tested. These soils were chosen from various localities in Wisconsin and must have included some soils acid to litmus, since Whitson and Weir (40) estimate that two-thirds of the soils of Wisconsin are acid. Christensen and Larsen (4) find that if Ashby’s solution is inocula- lated with a soil in need of lime, the brownish film usually produced by Azoto- bacter does not develop. They suggest this as a method of determining the need of a soil for lime.
Gimingham (11) describes several organisms capable of bringing about the formation of carbonates from calcium salts of organic acids. Hall and Miller (13) also report that calcium salts of organic acids are transformed to the carbonate by soil organisms, the organic acids being decomposed to form carbon dioxide and water. Drew (7) shows that marine bacteria precipitate calcium carbonate from sea water. He names the organism responsible for this re- action, Bacillus calcis. Kellerman and Smith (23) write that it is possible in the laboratory to produce calcium carbonate by three types of biological processes; by the action of ammonium carbonate on calcium sulfate; by the action of ammonium hydroxide on calcium acid carbonate, and by the de- composition of calcium salts of organic acids. They state that Drew’s organ-
BACTERIAL ACTIVITY AND LIME REQUIREMENT 437
ism is Pseudomonas calcis. This is a denitrifying organism which produces ammonia by the reduction of nitrates. Bear and Salter (2) show that the lime requirement (Veitch) of the West Virginia Agricultural Experiment Station fertility plots is less where the content of organic matter has been increased, and suggest that this decrease may have been due to the precipita- tion of calcium from solution by the humus in the soil, whereby it was pre- vented from being lost in the drainage water. This calcium might later be freed as the carbonate, as the decomposition of the organic matter was brought about by the soil organisms.
OBJECT OF THESE INVESTIGATIONS
In view of the fact that large areas of land are acid and that the distance from the supply of lime often makes the cost of applying large amounts of lime or limestone prohibitive, it was thought it might be desirable to consider ‘more carefully the possibilities of a system of acid agriculture as suggested by Coville (6). Since the problem of the economy of nitrogen and its availa- ability for the use of crops is largely a bacterial problem, it seemed important to study the relation of the reaction of the soil to the activities of the bacteria concerned in nitrogen accumulation and transformations. Recognizing the fact that plants do grow on soils which are acid to litmus, how are these plants supplied with nitrogen? We know that lime and limestone are valuable soil amendments, but might it not be possible that small applications of these materials would be relatively more effective in promoting the activities of the bacteria concerned in the nitrogen problem than large applications? If the B. radicicola of some legumes is more resistant to acidity than the B. radicicola growing on other legumes, might it not be possible to select legumes adapted to the reaction of the soil instead of adding lime to the soil to make the reaction suitable for the legumes we desire to grow? Even if nitrogen-fixing organisms are able to grow in acid soils, are they able to fix atmospheric nitrogen in such an environment? ‘To answer these questions, it was proposed to measure the activities of those bacteria concerned in the nitrogen economy of plants as influenced by various amounts of calcium carbonate applied to acid soils.
DEFINITION OF “LIME REQUIREMENT”
In the preceding discussion, a rather loose construction is given to the term “soil acidity.’ This is simply in accordance with precedents set by the various investigators whose work is reviewed. As a rule, an “acid” soil means a soil which changes blue litmus paper red. The “degree of acidity” of soils has no such definite meaning, consequently the investigations reported are not strictly comparable. The writer sees no reason to disagree with Truog (33) as to what “soil acidity” really is. Truog writes that acid silicates are the main cause of soil acidity in upland soils. His excellent review of this subject gives a select bibliography of the investigational work along this
438 FIRMAN E. BEAR
line. Truog (32) also writes that the acidity of soils may be conveniently divided into two classes, ‘‘active” and “latent” acidity. He states that “latent” acidity is undoubtedly much less injurious to plants than “‘active”’ acidity. He also shows the desirability of knowing the “avidity” of the active soil acids. Sharp and Hoagland (30) attempt to measure the lime requirement of soils by determining the hydrogen-ion concentration of the soil suspensions. The recent review of Clark and Lubs (5) of the literature on this subject, indicates that the hydrogen-ion concentration of the medium is the important factor to consider in the relationship between acidity and biological processes. The hydrogen-ion concentration of a soil in suspension in water is, however, not a measure of the amount of lime necessary to add to an acid soil to produce a neutral reaction of the soil. This is partly because of the slow solubility of the acid-forming constituents present in soils. .
At the time this investigation was begun, most of the recent work on soil acidity had not been published. The writer felt at that time that the most , satisfactory measure of the “lime requirement” of a soil was that obtained by the Veitch (34) method. Accordingly, this method was used in determining the quantitative need of the soils used for lime. It is interesting to note in this connection that when the two soils which were used most largely in these investigations had been treated with the quantity of calcium carbonate neces- sary to satisfy their lime requirements (Veitch) and had been mixed once each week for 12 weeks, they were found to be neutral to litmus paper.
HISTORY OF THE SOILS USED IN THESE EXPERIMENTS
A large part of the work reported has been done on samples of soil from two different localities belonging to different soil series. Both of these were acid in reaction, as will be shown later.
Soil I was secured from plot 18 of the West Virginia Agricultural Experi- ment Station farm. The soil is classified by the United States Bureau of Soils as Dekalb silt loam. It is a residual soil which has been formed by the disintegration of sandstone and greenish gray shales overlying the Pittsburg coal. The original timber was largely oak and chestnut with an occasional locust. The analysis of this soil is as follows:
Pounds per Element 2,000,000 by soil Nitrogenten. +o. Nes pe eee Fd Sens ee Atel ice aL oes Ee mR Ni Be 1,940 Jel oicyo nabs Ate a Soy OA Oe Oitmaa ec de hae neh tae a PIA a) 600 PO EASSHUIN AO aah a. DA eet vse Dace Pe AN NCR crea crete ote le tally cheat el a I 25,100 (CRIs OO) ale Mees Coc ERE GANA REERS Sibir OS Oe RECO MEREE Ene Caan EME tc 23,900 (CEI TUHIIT EE, BG ee ORES OO SEN REI Te OOS ee Ge NEN eo RRT sottes et CBRE ever rT 2,300 IY Reyes n ERVUUT OCT GOP RNS HiRIMOH ANA ORDS. S ca atlas ER Arr oie aaa om inte tenes eae Te 4,300 Galemummicarbonate requirement !(Vertch) iste. ase ea etieaneie dere ol 3,500
Plot 18 has not received any fertilizer, lime or manure since the beginning of the fertilizer tests in 1900. Only a partial record of the produce of this
BACTERIAL ACTIVITY AND LIME REQUIREMENT 439
plot is available. During a part of the time since 1900 a tile drain, which passed near this plot, was not working, and, since the yields of the plot were somewhat abnormal, no permanent records of the plot were kept. Later the record of the produce of this plot was continued. This record shows that plot 18 corresponds normally in productivity to plot 21, which also received no fertilizer, lime or manure. The sample of soil was chosen from plot 18 because its record was incomplete and any change due to the removal of a large sample of soil would not interfere with the plot experiments. Since 1900 the following crops have been grown on these plots; rye, 1900 and 1907; wheat, 1901 and 1914; clover, 1902, 1909, and 1915; corn, 1903, 1905, and 1912; cowpeas, 1904; potatoes, 1906; timothy, 1909, 1910, and 1911, and oats, 1913. Table 1 gives the records of the fertilizer treatment and total produce of all the plots up to and including 1915.
TABLE 1 Total amounts of fertilizers applied and total produce per acre from 1900 to 1915 on soil I
ae Se Re euosaaaee hp (Ca0) Ph steh te Seance pounds pounds pounds pounds tons pounds 49 NYP) K CaO. ..:. 4200 4200 1625 4500 120,605 MUM CaQ. (52 6<<.2- 4500 210 | 152,400 me tail @ HECK: 5)... : =. 38,600 210 | A CRY 0 ee ae aa 5500 36,615 a ose ie No 300 | Ash of 40 tons of manure until 1912 39,270 Bey Cheek! eo os 43,075 Ee Sc ee re 190 | 139,670 PAE AAU EST (ee ee 4200 4200 1625 117,910 BrinGheck:. 2 <.0;.... 42,170 1 al ee ae 4200 1625 76,995 OMNES Cs cho: see5 oes 4200 1625 52,215 Bd Checke ns... .... - | 39,480 i) 2 i en 4200 4200 95,940 SA. SUSI Sea eae 1625 41,565 Beal heck s...62....~. 36,845 SL AN Re te eee 4200 63,415 RISEN esere ers sic ae este ts 4200 41,195
N, indicates nitrate of soda; P, acid phosphate; K, sulfate of potash; M, manure.
Soil II was secured from the Ohio Agricultural Experiment Station farm at Wooster. This soil is classified by the Bureau of Soils as Wooster silt loam. It has been formed from the disintegration of sandstone and shales of the Mississippian period, under the influence of glacial action. The analysis of the soil used is as follows:
440 FIRMAN E. BEAR
Pounds per Element 2,000,000 of soil 1 SOIT cra BS Pe dd OAR 8 ah ee ae eS SAA, A LS PHOSpHUMUsh heidi dae) hee ise Wade Ohioek Serle cermnae sen es Hasse ate 664 Ro tascam: Sesto ae siew ys aiine ocaal ee MueOa atS Ne eToet eh Sis\s a ive o/S aus setae 34,000 (ESA BYOB RN crs eA RI Ate, =. RRA het ct een ren ORC EEE enaE NSN a cy 22,200 (Gea cette alae leis <i RS AN ae ce Mma eine hy Sip ices se rane 4,470 MVIAPRIESEUIN, ls tee Meals eh seeh ssa eters eee od shanties Sie Se Bin pdis Se t's. Mae 6,596 Calcium carbonate requirement (Veitch)................ cece eee 3,500
It will be observed that soil II has the same calcium-carbonate requirement as soil I.
Soil II has never received any fertilizer, lime or manure since the beginning of the fertilizer tests in 1893. Continuous records since that time have been kept on soil of the same history as this soil in a 5-year rotation experiment at the Wooster station. The rotation has been corn, oats, wheat, clover, and timothy. A summary of the effect of lime and fertilizers on this soil is given by Williams (41) in table 2. An experiment has also been in progress on this same type of soil which had been kept in a fair state of fertility by a good rotation and an occasional application of manure previous to the beginning of the experiment. The rotation since practiced has been corn, oats, and clover. The records of this experiment are shown in table 3. It will be seen by a study of tables 2 and 3, that both lime and acid phosphate are very effec- tive in increasing the yields of the crops grown in these two rotations. While lime is very efficient, it seems remarkable that such large yields of these crops can be produced by the use of acid phosphate alone on a soil which has a calcium-carbonate requirement of 3500 pounds per 2,000,000 pounds of soil.
The other samples of soil used in these experiments were Dekalb soils chosen from various localities in West Virginia. These soils vary greatly’ because of differences in the systems of management they have undergone. Analyses of these soils are shown in subsequent tables.
PLAN OF THESE EXPERIMENTS
Large samples of soils, acid to litmus, were secured, sent immediately to the laboratory, made to pass a 2-mm. sieve, and stored in large galvanized iron cans. From these cans soil was removed as needed. Careful analyses of the soils were made for the total amount of nitrogen, phosphorus, potassium, calcium, magnesium, and carbon. Lime-requirement determinations were made by the Veitch method as indicated above. Amounts of C. P. calcium carbonate varying from 250 pounds to 40,000 pounds per 2,000,000 pounds of soil were added to the soils. A study was made of the effects of these applications on: (a) the number of bacteria, (b) the rate of ammonification, (c) the rate of nitrification, (d) the fixation of nitrogen by non-symbiotic organisms, and (e) the development of B. radicicola of the soybean. All analyses were made according to the methods given by Bear and Salter (3).
BACTERIAL ACTIVITY AND LIME REQUIREMENT 441
The calcium carbonate was applied and mixed thoroughly with the soil, which was then placed in 1-gallon stone jars. Enough water was added to the soil to give it an optimum moisture content. Each week the soil was removed from the jars and mixed thoroughly and the loss of moisture, due to
TABLE 2 The effect of lime on the yields of crops on soil II
YIELD PER ACRE
Corn Oats Wheat Clover Timothy 1900-1915 1901-1915 1906-1916 | 1903-1916 | 1909-1916
PLOT TREATMENT
PA Webosplornus*. 00. ) sg%4s at 35.51/42. 32/39. 16/42 85/21. 48/25 . 17/1848/2680|3058|3810 8| Phosphorus,* potassium.... . 43 .95|51.08|42.62/46.38|22. 17|26. 38|2144|3166|3125|3881 11} Phosphorus*, potassium, ni- " BEMEIN Aas ort < 2.5 We tee ai dss 47 .67|55 .12}49.77/49 .71|31. 27/31 .86|2683|3388| 3445/4124 17} All three with less nitrogen
but more phosphorus*.. . . |47 .23/55.67/51.84|52.38|27 . 32/30. 85|2492|3598| 3364/4543
18| Barnyard manure......... ./56.31/61. 68/43 .62/44.93]/29. 51/32 .49|3448/4393/4525|5531
24| Same as 17 but nitrogen in sulfate of ammonia...... ./46.23/55.98/48 21/51. 36/24. 70/31. 26/2139|3544|3111|4409
26| Same as 17 but phosphorus i bone meal’... 2.55... 46 .01/51.17|46.37|46.81)27 . 7828 .65|2945|3772|3504|4585
29} Same as 17 but phosphorus in basic slag............ .|46.27|51. 69/47 .77|47 .85|29 . 76/28 .93|2981|337 137414306 Average unfertilized............. 26.48/32 .32|27 . 19|32 .08]12 .74|16 .09]1276]1841|2500|3069
* Phosphorus in the form of acid phosphate.
TABLE 3 The effect of lime and acid phosphate on soil II
CLOVER Sta aoe Awount CORN 9 YEARS OATS 9 YEARS Ricnans Grain Stover Grain Straw Hay
pounds pounds pounds pounds pounds pounds
PMOREGRUEMIZEN. «)./-.cra ccc & «rte 51.50 2759 44.94 1961 4074 CAeIMMOKIGE:........ 4... 1000 57.33 3149 47.53 2079 4580 Ground limestone......... 1780 54.84 2820 45.35 1876 4362 Acid phosphate........... 320 60.18 3056 46.16 1912 4277
evaporation, was restored. This was continued for 12 weeks in order that the soil microdrganisms should have time to adjust themselves to the changes in soil reaction. At the end of that time, the determinations of nitrifying power, ammonifying power, etc., were made. These determinations required about one-half of the soil.
442 FIRMAN E. BEAR
Since the analyses showed that these soils were very deficient in total phosphorus, a thing which is commonly true of acid soils, it seemed advisable to apply phosphorus in a readily available form in order to remove it from being a possible limiting factor in the various bacterial activities studied. Accord- ingly, 0.2 per cent of mono-calcium phosphate, equivalent to 1000 pounds of phosphorus per 2,000,000 pounds of soil, was added, the moisture content was again restored, and the mixing was continued for another period of 12 weeks. At the end of this time, the above determinations were repeated. In some of the later experiments the calcium carbonate was added just previous to the time of studying the rate of nitrification, ammonification, etc.
THE EFFECT OF CALCIUM CARBONATE ON THE NUMBER OF BACTERIA
Soils I and II were used in these experiments, after they had received the various applications of calcium carbonate and had been mixed thoroughly each week for 12 weeks, as previously outlined. Plate counts of the number of microdrganisms were made at the end of the 12-week period. After the 0.2 per cent of mono-calcium phosphate had been added and mixed with the remainder of the soil each week for a second 12 weeks, plate counts were again made. Aliquots of the soil suspension were plated on Heyden agar. The plates were incubated at room temperature and counts were made at the end of 6 days. Table 4 shows the results of these counts. Each figure represents the average of four plates.
As might be expected, the greatest relative change in the number of bac- teria occurred after the neutral point had been passed. This was true in
TABLE 4
The effect of calcium carbonate on the number of bacteria
BACTERIA PER GRAM OF SOIL CALCIUM CARBONATE PER 2,000,000
POUNDS OF SOIL Soil I without Soil I with Soil II without Soil II with phosphorus phosphorus phosphorus phosphorus pounds 0 3,341,000 4,150,000 3,503,000 3,438,000 250 4,127,000 4,320,000 3,418,000 3,536,000 500 3,537,000 3,540,000 4,614,000 4,421,000 ¥,000 3,439,000 2,750,000 3,781,000 5,207,000 2,000 3,930,000 2,520,000 4,472,000 5,781,000 3,000 4,127,000 4,090,000 4,919,000 5,683,000 Neutral point (Veitch method)
4,000 4,422,000 5,820,000 7,348,000 10,005,000 5,000 5,306,000 7,700,000 9,741,000 17,392,000 7,500 5,601,000 7,070,000 15,827,000 14,297,000 10,000 3,341,000 6,680,000 14,973,000 7,959,000 20,000 6,682,000 10,750,000 14,892,000 3,635,000 40,000 9,335,000 13,050,000 18,199,000 9,826,000
BACTERIAL ACTIVITY AND LIME REQUIREMENT 443
both soils, as shown in figure 1. The 4000 and 5000-pound applications of calcium carbonate resulted in relatively large increases in numbers. Additions of calcium carbonate in excess of 7500 pounds per 2,000,000 pounds of soil gave somewhat uncertain results. There was a decrease in every case ac- companying an application of 10,000 pounds as compared with 7500 pounds of calcium carbonate. The 20,000 and 40,000-pound applications brought about marked increases in numbers. These fluctuations were probably due to the adjustment of the soil reaction to the point where it was more suitable to the requirements of some forms which developed in vast numbers under this optimum soil reaction. It seems quite evident that the application of calcium carbonate caused decided changes in the number of bacteria in these soils. The maximum increases in numbers had.apparently not been reached in three of the four cases by applications of 40,000 pounds of calcium carbonate per 2,000,000 pounds of soil. Similar trials with calcium oxide produced marked decreases in the number of bacteria in these soils following the larger appli- cations. This was probably due to the partial sterilizing action of calcium oxide previously referred to.
THE EFFECT OF CALCIUM CARBONATE ON THE RATE OF AMMONIFICATION
Soils I and II, after the treatments with calcium carbonate and mono- calcium phosphate previously referred to, were used in the experiments on ammonification. The source of nitrogen was Hammarsten’s casein. Enough casein was added to supply 160 mgm. of nitrogen per 100 gm. of soil. The soil was then given an optimum moisture content and incubated in tumblers for 3 days at room temperature, after which analyses were made for ammonia by distillation with magnesium oxide. Each figure given in table 5 represents the average of two determinations which checked usually within less than 1 mgm. per 100 gm. of soil. Other determinations, not reported in this paper, were made in which only 40 mgm. of nitrogen were added, with very satis- factory results. The author believes that 160 mgm. of nitrogen per 100 gm. of soil are likely to produce abnormal conditions in a soil, although in most of the ammonification experiments reported in the literature even larger amounts of nitrogen were supplied.
The greatest relative increase in the rate of ammonification of casein, per unit of calcium carbonate applied, occurred with applications of 2000 pounds of calcium carbonate per 2,000,000 pounds of soil,as shown in figure 2. There was no marked increase in ammonification as the neutral point was passed. As previously shown, this was also the case in the number of bacteria. Appli- cations of 250 pounds of calcium carbonate per 2,000,000 pounds of soil had a tendency to cause a decrease in the rate of ammonification. Applications of calcium carbonate in excess of 5000 pounds caused only a slight increase in the amount of ammonia produced. The 20,000 and 40,000-pound applications caused slight decreases in ammonia in several cases. There was
444 FIRMAN E. BEAR
apparently no definite correlation between the number of bacteria and the amount of ammonia produced, although in general, increased amounts of calcium carbonate resulted in larger numbers of bacteria and more rapid ammonification.
THE EFFECT OF CALCIUM CARBONATE ON THE RATE OF NITRIFICATION
The effect of calcium carbonate on the rate of nitrification in soils I and II is shown in table 6. All figures in this and in succeeding tables of nitrification
TABLE 5
The effect of calcium carbonate on the rate of ammonification of casein*
NITROGEN AS AMMONIA PER 100 GM. OF SOIL CALCIUM
CARBONATE PER
coalee sid a SoilIwith | Soil It Soil IT | Soil ILwith | Soil IL with
AD wspolshio oer Bi aaa phosphorus Ease ts phosphorus phosphorus pounds mem mgm mgm. mgm mgm mem 0 72.40 60.70 38.85 22.89 29.51 39.20 250 71.00 61.00 40.25 22205 25.55 37 .38 500 72.80 59.60 45 .36 29.05 28.00 37.80 1,000 75.40 62.40 48 .02 29.40 28.63 44.31 2,000 78.50 68.00 57.54 41.65 43.40 55.44 3,000 79.00 70.00 56.00 44.59 45 .36 ST 1S
Neutral point (Veitch method)
4,000 78.40 70.60 60.41 45.85 56.84 63.00 5,000 76.30 70.80 67.27 57.12 64.61 71.54 7,500 85.30 74.80 71.40 57.05 52.29 67.97 10,000 83.60 74.80 74.48 62.79 63.07 71 61 20,000 85.40 77.80 77.40 60.76 55.27 68.81 40,000 87.50 77.60 U1 42 61.25 51.61 59.36
* The results in each vertical column were obtained on the same day. Fluctuations in the temperature in the room are responsible for some of the differences observed in horizontal columns. .
7 Four-day periods of incubation.
represent averages of two determinations. As a rule, the duplicates agreed within less than 0.1 mgm. per 100 gm. of soil. Accordingly, only averages are reported.
These soils were treated with calcium carbonate in varying amounts and with mono-calcium phosphate as previously outlined. At the end of the 12- week periods, samples of these soils of 100 gm. each were placed in 1000-cc. Erlenmeyer flasks for the nitrification experiments. To each flask were added 20 mgm. of nitrogen in the form of either ammonium sulfate or ammo- nium carbonate. After adding water to the optimum content, the soils were incubated for 21 days at room temperature, after which the nitrate determina- tions were made by the phenol-disulphonic acid method.
BACTERIAL ACTIVITY AND LIME REQUIREMENT 445
A study of table 6 and figure 3 shows that the addition of calcium carbonate is followed by an increased nitrification which correlates almost directly with the increased application of calcium carbonate. This correlation holds fairly well in every case with applications up to 5000 pounds per 2,000,000 pounds of soil. There is no sudden break in the correlation as the neutral point is passed. Applications of calcium carbonate in excess of 5000 pounds are followed by increased nitrification, although the curve of increase begins to incline more toward the horizontal. In half of the experiments the curve was still ascending with applications of 40,000 pounds of calcium carbonate.
TABLE 6 * The effect of caictum carbonate on the rate of nitrification NITROGEN AS NITRATE PER 100 GM. OF SOIL
CALCIUM CARBON-
Source of nitrogen, ammonium sulfate Source of nitrogen, ammonium carbonate.
ATE PER 2,000,000 PouUNDS
OF SOIL Soil I* Soil I Soil II Soil II Soil I Soil I Soil II Soil II
without with without with without with without with
phos- phos- phos- phos- phos- phos- phos- phos-
phorus phorus phorus phorus phorus phorus phorus phorus
pounds mgm. mgm. mgm. mgm. mgm. mgm. mgm. mgm. 0 1.08 4.06 5.28 6.07 1.38 22 5.29 7.50 250 1.38 4.32 4.39 5.34 1.93 8.24 5.00 8.00 500 1.43 4.60 4.40 6.35 aoe 8.42 5.42 8.40 1,000 1.82 5.24 6.15 Onis Bal 9.52 6.60 8.55 2,000 2.29 6.38 8.50 8.73 3.01 12.42 9.03 155 3,000 2.96 9.34 10.48 10.43 OND 15.30 10.04 12250
Neutral point (Veitch method)
4,000 3.18 11.92 15.74 122509) 33.28 17.50 11.87 R12 5,000 3.44 13.86 15.96 15.00} 4.11 18.00 15a 18.75 7,500 3.48 16.37 18.18 15.38 | 4.69 19.00 17.28 16.35 10,000 4.44 19.35 20.98 15.98 | 4.30 20.00 | 20.30 16.16 20,000 4.00 20.45 22.87 16.00 | 4.32 20.96 19.80 16.00 40,000 4.20 2259 19.88 15.00} 5.18 23.30 | 20.57 15.00
* An error was made in calculating the optimum moisture content and the soil in this experiment was too dry.
This is directly contrary to the work of Scales previously referred to, which indicated that 50 per cent of the amount of calcium carbonate necessary to supply the lime requirement of the soil is sufficient to attain the maxi- mum rate of nitrification. Additional amounts are reported to have acted injuriously.
The marked increase in the rate of ammonification observed with the addition of 2000 pounds as compared to 1000 pounds of calcium carbonate per 2,000,000 pounds of soil was not followed by a coresponding increase in the nitrification. No correlation was found between the increased number of bacteria in the soil and the rate of nitrification except that in general the
446 . FIRMAN E. BEAR
application of increased amounts of calcium carbonate caused an upward tendency in the number of bacteria, as well as in the rate of nitrification. Since the agar plate method is not designed to include the nitrifying bacteria, no data are available as to the actual number of nitrifying organisms which were present in the soils following the applications of varying amounts of calcium carbonate.
EFFECT OF CALCIUM CARBONATE ON THE RATE OF NITROGEN FIXATION BY NON-SYMBIOTIC SOIL ORGANISMS
Samples I and II were employed again in these experiments after they had been treated as previously described. Shallow dishes having a depth of about 3 inches and a capacity of 400 gm. of soil were used for this work. Soil
TABLE 7
The effect of calcium carbonate on nitrogen fixation by non-symbiotic soil organisms
CALCIUM CARBONATE PER NITROGEN FIXED PER 100 GM. OF SOIL
, ,
SOUNDS C2 SOs Soil I without phosphorus |Soil II without phosphorus} Soil II with phosphorus
pounds mgm. mgm. mgm. 0 0 250 0 500 0. 1,000 1 2,000 0 3,000 0)
4,000 0.1 2.8 12 5,000 1.8 4.1 1207 7,500 2.0 6.0 iss 10,000 175 5.8 10.1 20,000 0.1 4.5 12.6 40,000 1.4 44 9.9
from the various pots to which the calcium carbonate and mono-calcium phosphate had been applied was placed in the dishes and mixed thoroughly with 2 per cent of mannit. Optimum moisture conditions were secured and maintained as nearly as possible by adding water twice daily to restore that lost by evaporation. The soils were incubated 21 days at room temperature. After thorough drying, the entire samples were pulverized to pass a 100-mesh sieve and the total nitrogen was determined in triplicate. The triplicates agreed usually within 0.05 mgm. of nitrogen on 10-gm. samples.
From a study of table 7, it appears evident that both calcium carbonate and mono-calcium phosphate were essential to the highest fixation of nitrogen. The mono-calcium phosphate had such a marked effect in increasing the nitro- gen-fixing power of soil II, as shown in figure 4, that it would seem that phos-
BACTERIAL ACTIVITY AND LIME REQUIREMENT 447
phorus was equally as important as lime for the nitrogen-fixing organisms in this soil. The largest relative increase in nitrogen fixation followed an applica- tion of 3000 pounds of calcium carbonate per 2,000,000 of soil when accom- panied by the use of mono-calcium phosphate. Heavier applications of calcium carbonate caused an increase in nitrogen fixation until as much as 10,000 pounds per 2,000,000 pounds of soil had been applied. This amount and heavier applications caused a decrease in nitrogen fixation. Apparently phosphorus was a limiting factor in nitrogen fixation in soil I, although time did not permit an experimental test of this point. The good effects resulting from the use of acid phosphate on these soils under field conditions may be due in part to this increased nitrogen fixation accompanying its use. This again is indicated by the analyses of the West Virginia Station fertility plots from which the author and others have shown that the plot receiving acid phosphate and sulfate of potash has accumulated 1173 pounds of nitrogen per acre during the last 15 years which could not be accounted for except by nitrogen fixation from the air. The evidence shown in table 7 indicates that calcium carbonate is necessary in addition to the phosphorus for the most effective nitrogen fixation.
Following the suggestion of Christensen and Larsen (4), soil from each of the pots to which varying amounts of calcium carbonate had been applied, was used to inoculate Ashby’s solution in order to study the relation between - the film development on the surface of the liquid and the lime requirement of the soil. The brownish film was very well developed in the flasks inoculated with soil which contained an amount of calcium carbonate in excess of the requirement of the soil and practically disappeared as the quantity of calcium carbonate applied was reduced below the amount necessary to satisfy the requirement of the soil. The development of brown pigment is apparently closely related to the amount of lime in the soil and may be used as an index of the need of lime by the soil. Other experiments which will be discussed later indicated, however, that nitrogen fixation in Ashby’s solution may take place when the solution is inoculated with soils having calcium-carbonate requirements as high as 4600 pounds per 2,000,000 pounds of soil. Apparently, the lack of development of the brownish film is not accompanied by the loss of ability to fix nitrogen, since no soil was found which did not show nitrogen fixation when inoculated into Ashby’s solution and allowed to stand for 21 days at room temperature.
THE EFFECT OF CALCIUM CARBONATE ON THE FIXATION OF NITROGEN BY B. RADICICOLA OF THE SOYBEAN (SOJA MAX PIPER)
Soils I and II were again used in these experiments. One-gallon pots were filled with these soils and the calcium carbonate was added. Each pot re- ceived an application of 0.2 per cent of mono-calcium phosphate. The pots were planted to soybeans, the beans having been previously inoculated with
448 | FIRMAN E. BEAR
B. radicicola. Six beans were planted in each pot and later thinned to three per pot. After the beans had reached the stage where pods were formed, they were harvested, and records were taken of their green and dry weight, the number of nodules, the dry weight of nodules and the milligrams of nitrogen in the roots, tops and nodules. One crop was harvested from each pot during the summer of 1915 and another crop during the summer of 1916. The records are shown in tables 8 and 9.
The number of nodules had a tendency to increase slightly with small appli- cations of calcium carbonate. Applications of more than 3000 pounds of calcium carbonate per 2,000,000 of soil caused a decrease in the number of nodules. This decrease was proportional to the amount of calcium carbonate applied. The dry weight of nodules was also decreased with large applications of cal- cium carbonate. The rate of decrease in dry weight of nodules with increased amounts of calcium carbonate was more marked than the rate of decrease in the number of nodules. The amount of nitrogen in the nodules was almost directly correlated with the dry weight of nodules, and decreased ‘with addi- tional quantities of calcium carbonate. It will be noticed that in both cases the dry weight and total nitrogen of both stems and roots had a tendency to increase with small applications of calcium carbonate, but that applications in excess of 2000 pounds per 2,000,000 of soil had a tendency to cause a de- crease in dry weight and total nitrogen of the stems and roots.
The total nitrogen fixed by soils I and II during the two years in which the two crops of soybeans were grown was determined. Analyses of the soil were made before and after the beans were grown. ‘The difference in the nitrogen content of the soil at these two periods plus the nitrogen removed in the nodules, stems and roots, after subtracting the nitrogen content of the seed and water used in watering the plants, represents the nitrogen secured from the air.
The total nitrogen fixed in two years per 2,000,000 pounds of soil, as shown in the last columns of tables 8 and 9, indicate that soil II has had a more active nitrogen-fixing flora than soil I. In so far as the chemical composition is concerned, the two soils correspond fairly well, as will be found by referring to the analyses of these two soils previously shown. By referring again to table 7, showing the rate of nitrogen fixation by Azotobacter, it will be seen that soil II was much more active in this respect than soil I. It is possible that a greater part of the nitrogen accumulated in soil II during the growing of the legumes was fixed in the soil through the agency of the non-symbiotic organisms. The nitrogen fixation had a tendency to decrease with applica- tions of calcium carbonate in excess of 2000 pounds per 2,000,000 pounds of soil, although the lime requirement of both soils indicated a need of 3500 pounds. Apparently, with increased applications of calcium carbonate the rate of nitrification was so high, as indicated in table 6, that the soybeans were able to secure a greater part of their nitrogen in the form of nitrates. Large numbers of B. radicicola were present in all the pots whether treated with
BACTERIAL ACTIVITY AND LIME REQUIREMENT 449
TABLE 8
The effect of calcium carbonate on nitrogen fixation by B. radicicola of the soybean in soil I
CALCIUM NODULES STEMS ROOTS SOIL CARBONATE : Nit PER Nitr * rogen POT | 2,000,000 |NUMPER) | Total | p, | Total | p,. | Total | gen in Pe fixed per ees, os weight ae weight gue weight ea gee nd per Sincdeet per pot} P° soil pounds mgm. mgm grams | mgm. | grams mgm. | grams | grams pounds 1 0 HASH EOSSe ie SL 13.2 | 348] 4.3} 40 |3.0009)2.8985 93 2 250 67 | 887 | 39 14.2 | 377 | 5.1} 42 |3.0039/2,8675 98 3 500 88 | 749 | 33 14.3 | 368} 4.4} 39 |3.0039/2.8985 107 5 2,000 100 | 1017 | 47 17.3 | 464] 3.7] 35 13.0039/2.8272 130 6 3,000 F207 560) |} (27 14.9} 303 | 3.4] 38 |3.0039|2.7900 +8 Neutral point (Veitch method) 7 4,000 65; |)-317 16 11.9 | 342] 2.9 | 37 |3.0039|2.7683 —6 8 5,000 719) 2537 26 14.1 363 | 3.6] 39 |3.0039/2.8923 94 9 7,500 84 | 464] 22 13.9] 426] 3.8 | 46 |3.0039/2.7218 23 10; 10,000 66) 212 11 1152) | S425) 364) 52) 1310039276660 —60 11} 20,000 AS) 220%) 13 LORS eZOSe GaSe 58 |3.0039}2.6815 —87 12} 40,000 yp |) Pl 13 12.2 | 383 | 3.4] 50 |3.0039|2.6505 —56
0.1727 gm. of nitrogen in the soybeans planted. 0.0043 gm. of nitrogen in the water used in watering the soybeans.
TABLE 9
The effect of calcium carbonate on nitrogen fixation by B. radicicola of the soybean in soil II
CALCIUM
ROOTS
SOIL
CARBONATE por | 000.0 00 | NUMBER POUNDS OF SOIL pounds 1 0 79 3 500 75 6 3,000 | 148 8 5,000 fal 9 7,500 85 10} 10,000 66 11| 20,000 54 12} 40,000 33
STEMS Total
D ‘ Weiaht pr
grams | mgm
14.8 394 152 440 1325 371
Neutral point (Veitch method)
NODULES Total : tro- weight - 4 mgm. | mgm. 781 38 744} 37 577 29 330 18 Z5iL 13 207 12 124 5 126 7
12.3 | cp LS 14.4
338 370 335 341 393
0.1727 gm. of nitrogen in the soybeans planted. 0.0043 gm. of nitrogen in the water used in watering the soybeans.
\
SOIL SCIENCE, VOL. Iv, NO. 6
grams 2.6181)3. 2008 2.6181|3. 1860 2.6181)3.1418
grams
2.6181]3. 1358 2.6181|3.1270 2.6181/3.0238 2.6181|2.9648 2.6181|2.9205
Nitrogen fixed per 2,000,000 pounds of soil pounds 570 587 $11
476 490 408 377 265
450 FIRMAN E. BEAR
calcium carbonate or not, although quantitative determinations were not made of their numbers.
THE EFFECT OF CALCIUM CARBONATE ON SOYBEANS UNDER FIELD CONDITIONS
In order to determine whether soybean yields are increased by the use of calcium carbonate on soil I under field conditions, it was decided to grow soybeans on the fertility plots of the station farm during the summer of 1916. Three varieties of soybeans were sown in rows across the plots and cultivated — during the growing season. One-half of each plot received an application of calcium carbonate at the rate of 2 tons per acre in the form of ground lime- stone. The yields of hay produced are given in table 10. The previous crop records and the analyses of the soils of these plots are given in tables 1 and 11.
TABLE 10 Effect of ground limestone on yield of soybean hay on soil I
Ba ercsha YIELD OF HAY PER ACRE PLOTS TREATMENT REQUIREMENT pepsi sa ge ted igh No limestone Limestone
pounds pounds pounds per cent 19 INE OK iCa@ike wrecees - 0 5270 5400 +2 20 IMI OAO ce oe ee he See 0 6390 6850 +7 21 (hecklers ie han aie 2800 1605 1400 —13 De. O10 Lys aoe ite) eee eA ae 0 1920 2430 +26 26 IY Cae AAR bey eon eee ae 2800 7150 7360 +3 26 IN ul eee Se «on fate S eRBO-e Nee 3200 5300 5370 +1 28 | Dep are te Bry Sf A Pa ea 3600 2820 4690 +66 29 UNE Kee Steere actegate tres chat tn 3400 1285 2280 +77 31 Inia dS dates 1s Saad Gy & Eeneae 3200 3375 4460 +32 32 1 eee, ca Ad SO ocr arte iP 3600 1495 1995 +34 34 | EO reece oS ae ae 3400 3285 4105 +25 35 ae TS a ead lc orice Bictcn en ae 3400 1220 1705 +40 aT ELE Ss oy Gee oan ac SERRE IO CECA A ess IE 3426 4004 +17
From a study of the plots and the crop records, it would seem that the use of 2 tons of limestone per acre did not give sufficient increase in yield to justify the conclusion that soybeans will not grow well except on soils which have had their lime requirement satisfied. On plots 25 and 26, the soils of both of which have rather high calcium-carbonate requirements, but which also contain a fairly high content of nitrogen, the yield of soybeans was little affected by the limestone. This might mean that more nitrogen was secured from the soil on these plots and for this reason the crop was larger. However, the nodules were plentiful on the roots of the soybeans on plots 25 and 26 and, therefore, we could assume that nitrogen fixation from the air was taking place.
BACTERIAL ACTIVITY AND LIME REQUIREMENT 451
EFFECT OF CALCIUM CARBONATE ON THE BACTERIAL ACTIVITIES OF DEKALB SOILS HAVING VARYING LIME REQUIREMENTS
A large number of samples of acid soils all belonging to the Dekalb series were chosen from various parts of West Virginia and sent to the laboratory. From this number 12 samples were chosen which had calcium-carbonate requirements varying from 400 to 4600 pounds per 2,000,000 pounds of soil. The analyses of these soils are shown in table 11. These soils differ mostly because of the different systems of management practiced by the men who have farmed them since the areas from which the samples were chosen were cleared from the forest. Many of these areas had been farmed for from seventy-five to one-hundred years and others had not been farmed for more than a few years.
TABLE 11 Analyses of soils of table 12 Ra we DES ESS a a RB SIU RA Gh rs | al Lac9
POUNDS PER 2,000,000 PoUNDs OF SOIL
Nitrogen Phosphorus | Carbon Calcium-carbonate
requirement pounds pounds pounds pounds III 3870 1203 41,420 400 IV 1904 586 21,790 1000 V 1669 680 17,600 1200 VI 3374 697 47,230 1400 VII 3142 902 32,140 1600 VII 2042 1216 20,280 2000 Ix 2602 662 32,450 2200 x 4142 1135 48,680 2600 XI 3384 660 39,490 2800 XII 2750 706 32,140 3200 XIII 1960 608 21,900 3800 XIV 3124 753 48,280 4600
The rates of nitrification, ammonification, and nitrogen fixation were studied in an attempt to determine whether there was any relation between the activi- ties of the soil organisms and the calcium-carbonate requirements of these soils.
In nitrification studies 100 gm. of soil to which varying amounts of calcium carbonate had been added were placed in 1000-cc. Erlenmeyer flasks and incubated with optimum moisture content at room temperature for 21 days, using ammonium sulfate as the source of nitrogen, adding a sufficient amount to supply 20 mgm. of nitrogen per 100 gm. of soil. In ammonification studies 100-gm. samples of soil were used and varying amounts of calcium carbonate were added as in the nitrification tests. Casein was used as the source of nitrogen, 160 mgm. of nitrogen being added to 100 gm. of soil. The soil was incubated in tumblers at optimum moisture content for 3 days and the am-
452 FIRMAN E. BEAR
monia determined by distillation with magnesium oxide. Nitrogen-fixation tests were carried on by placing 10 gm. of soil in 100 cc. of Ashby’s solution in 800-cc. Kjeldahl flasks for a period of 21 days at room temperature. To one set of flasks enough calcium-carbonate was added to be equivalent to 10,000 pounds per 2,000,000 pounds of soil. At the end of 21 days the total nitrogen was determined. All of the determinations on nitrification, ammoni- fication, and nitrogen fixation were performed in duplicate and these dupli- cates as a rule checked very closely. The results of these experiments are tabulated in table 12.
In general the highest rates of ammonification occurred with soils having the lowest calcium-carbonate requirements. The applications of 2000 pounds and 5000 pounds of calcium-carbonate brought about marked increased in the rate of ammonification. Applications of 10,000 pounds of calcium car- bonate per 2,000,000 pounds of soil caused a decreased ammonification except in soils XIII and XIV, which had calcium-carbonate requirements of 3800 and 4600 pounds, respectively. Apparently the application of 10,000 pounds of calcium carbonate per 2,000,000 of soil on soils having calcium-carbonate requirements of less than 3800 pounds is injurious to ammonifying organisms.
There was no very definite correlation between the rate of nitrification of ammonium sulfate and the calcium-carbonate requirement of the soils. In general, the soils having high calcium-carbonate requirements had a very low nitrifying power. With soils having calcium-carbonate requirements in excess of 2200 pounds per 2,000,000 pounds of soil, the nitrifying organisms did not become markedly active even with large applications of calcium car- bonate. Either the nitrifying organisms were almost entirely absent or had become very inactive because of the unfavorableness of the medium in which they were living.
There was no very marked correlation between the calcium-carbonate requirement of these soils and the nitrogen-fixing power of the soil organisms in Ashby’s solution. Soil XIV, having a calcium-carbonate requirement of 4600 pounds, was able to fix nitrogen to the extent of 2.9 mgm. per 100 cc. of Ashby’s solution in 21 days. The rate of nitrogen fixation was increased in every case by the addition of calcium carbonate, but the effect was more marked on soils having a high requirement than in soils having a low calcium- carbonate requirement. It seems remarkable, however, that nitrogen fixa- tion took place in all cases even though some of the soils had very high lime requirements.
EFFECT OF FERTILIZERS ON THE BACTERIAL ACTIVITIES OF SOILS
These experiments were conducted in order to determine what effect differ- ences in the fertilizer treatments of the same soil would have on the bacterial activities in the soil. Samples of soil were chosen from 12 plots of the fertilizer series of the fertility plots on the West Virginia station, some of which differ considerably because of the fertilizer applications they have received during the last 15 years. Records of the treatments of the soil on these plots have
BACTERIAL ACTIVITY AND LIME REQUIREMENT
TABLE 12
453
The effect of calcium carbonate on the activities of soil bacteria in Dekalb soils having varying
lime requirements
CALCIUM- ha viae NITROGEN PER 100 GM. OF SOIL CARBONATE
SORE ae NOO OGG meee "000,000 Nitrogen as _ Nitrogen as POUNDS OF SOIL POUNDS OF SOIL auiregy fiom pele biel ar ay seaees es ae Ry tprmared on. mgm. relative acne ee 0 68.9 | 100 8.0 100 2,000 1055: |) @ftt 12.6 158 oe =a 5,000 | 87.6| 127 | 12.5| 156 10,000 82.4] 118 9.4 118 ( 0 67.1 | 100 4.4} 100 2,000 Bio) Tae 8.4 191 Kea 2 ye 5,000 86.4] 129 13.5 307 10,000 74.6 | 111 14.0|.. 318 ( 0 68.9 | 100 1.3} 100 2,000 80.5 | 117 7.8 600 x — 5,000 | 78.1] 114 | 12.0] 969 10,000 72.6 | 105 16.0 | 123% 0 73.5 | 100 5.8 100 2,000 82) 20 tig 23 126 _ 2a00 5,000 | 86.9] 118 | 8.0] 138 10,000 Sh7 99 12:6)" 207 ( 0 56.8 | 100 1S 100 2,000 Pres h 136 S10. 200 — — 5000 | 91.8| 161 | 5.71 380 10,000 B75 |, 154 16.0 | . 1067 f 0 67.0 | 100 0.4} 100 ] 2,000 76.2 | 114 1.5.5 aie - = 5000 | 96.6] 129 | 8.01 2000 10,000 87.3 | 130 15:7 },(:3025 ( . 0 69.6 | 100 0.8 100 2,000 §3.1'| 119 5 638 1x a 5,000 | 90.1] 129 | 9.8] 1225 10,000 82.7) 819 4.6| 575 ( 0 62.9 | 100 27 t= 100 2,000 75.1%) 119 4.8 144 a ge 5000 | 92.2| 147 | 5.2] 156 10,000 91.8 | 146 13.0] 390 ( 0 51.6 | 100 0.8 100 2,000 68.3 | 132 eo 275 cd — 5,000 $8.9 a7 ey 213 10,000 92.2 | 159 Ae | 263
NITROGEN FIXED IN
100 cc. oF ASHBY’S SOLUTION
mgm.
7.6
8.7
4.2
5.6
EA |
4.1
4.4
5.6
4.9
5.9
af
6.5
Py
4.7
3.4
4.8
2.3
Ge
relative
100
114
139
454
FIRMAN E. BEAR
TABLE 12—(Continued)
SOIL
CALCIUM- CARBONATE RE- SATION, PER 2,000,000
POUNDS OF SOIL
NITROGEN PER 100 GM. OF SOIL
CARBONATE APPLIED oe SEN PER 2,000,000 POUNDS OF SOIL
Nitrogen as ammonia from casein
_ Nitrogen as nitrates from am- monium sulfate
a NN |
relative mgm.
XIII
XIV
mgm. 49.6 70.5 93.0 89.5
46.0 62.1 103 81.3
100 142 187 18Q
100 135 166 177
100 152 241 288
QR Qe
oro oOo
relative 100 350 500 800
100 273 410 345
100 233 367 233
NITROGEN FIXED IN 100 cc. oF ASHBY’S SOLUTION
mgm. relative 4.4 100 5.6 127 5.4 100 Heil 131 2.9 100 4.4 152
been given in table 1 previously referred to.
PLOT
19 20 21 De 25 26 28 29 31 32 34 35
TABLE 13
Analyses of the soil on the vari- ous plots were made and are recorded in table 13.
The studies in nitrification, ammonification, and nitrogen fixation were conducted in the same manner as previously mentioned in the discussion of the 12 soils of the Dekalb series with varying calcium-catbonate requirements. It will be remembered that the soil of the fertility plots is also Dekalb soil. The records of these experiments are shown in table 14.
Analyses of soils of table 14 |
TREATMENT
POUNDS PER 2,000,000 POUNDS OF SOIL,
Nitrogen Phosphorus Carbon pounds pounds pounds 2130 765 " 24,500 2700 1045 32,500 1830 590 21,200 1750 510 19,400 3240 1220 36,800 2665 900 30,400 2280 850 26,000 2290 640 27,000 2395 880 28,000 2310 740 29,200 2300 885 28,200 2100 620 28,800
Calcium- carbonate requirement
pounds
N indicates nitrate of soda; P, acid phosphate; K, sulfate of potash; M, manure.
BACTERIAL ACTIVITY AND LIME REQUIREMENT
TABLE 14
455
The effect of calciwm carbonate on the activities of soil bacteria in Dekalb soils which have received
varying fertilizer treatments
SSS SSS a eee eee
CALCIUM-
REQUIRE- TREATMENT
MENT PER
2,000,000
SOIL
pounds mK CaO... .: 0 INE CaO:.. 0
RON in cic a bane 2,800
Re. Sos tet 2-800 Mb aKen”.,,.+ 3,200 1 ee +} 3,600 [Sas ee ee 3,400
MAE coc lem esi 200
| | | | | | | | |
CALCIUM CARBONATE | CARBONATE APPLIED PER 2,000,000 POUNDS OF | POUNDS OF
SOIL
pounds 0 2,000 5,000 10,000
0 2,000 5,000
10,000
NITROGEN PER 100 GM. OF SOIL
Nitrogen
ammonia from
casein
mgm. relative 78.3 100 80.1 102 78.3 100 77.4 99 87.8 100 89.5 102 87.0 99 84.3 06 60.6 100 65.8 108 (Seat 2129 79.7 131 71.8 100 452 105 82.9 115 S257 115 i oa 100 153 105 90.5 126 90.0 125 70.1 100 80.3 114 85.5 122 86.6 126 58.4 100 66.2 109 76.3 131 82.4 141 56.6 100 65.9 116 75).5 133 81.8 144 60.9 100 68.7 113 82.9 136 85.2 140
as
Nitrogen as
nitrates from am-
monium sulfate
mgm. 15:3
_ oo OoWwonm
CRO”
— won = oOnwW Pp
ont RR DOMN
lel moO Ue Co “I CO CO
— nN mw ont
relative
100 121 127 133
100 123 129 126
100 450 983 1275
100 187 215 259
100 186 243 321
100 241 321 475
100 301 601 928
100 320 467 667
100 322 504 701
NITROGEN FIXED IN 100 cc. oF ASHBY’S SOLUTION
mgm.
a0.
t35
6.2
6.4
relative
100
134
100
113
100
456 FIRMAN E. BEAR
TABLE 14—(Continued)
ee ——————— 00€0—
CALCIUM- | CALCIUM NITROGEN PER 100 GM. OF SOIL CARBONATE | CARBONATE Be SCO Cy RTE | OPT a SG rs oe IN LADESRUY SSID ie ale Nitrogen as Nitrogen as ee 2,000,000 2,000,000 : A A SOLUTION rounps or | rooms or | S™monium irom | nitrates to oe pounds Pe pnnies| mgm. relative | mgm. relative mgm. relative eal 0 47.9 100 ial 100 7.4 100 K 2,000 57.4 120 2 ai Hid 7b a Ta 5,000 Tait 158 5.0 454 10, 000 84.0 174 6.6 600 7.6 103 49.5 100 sil 100 5.3 100 D5, a 65.0 131 3.4 301 eS ope el 5,000 79.4 160 7.0 636 10,000 84.3 170 8.5 We. 7.6 143 ( 0 5002 100 100 3.8 100 Nene oy ae —
5,000 78.6 | 156
3,400 2,000 64.9 | 129 10,000 $3.2 || 165
ION e Ww UO w S f=)
663 | 6.9 182
Nitrification of ammonium sulfate was not very active in these soils except on the plots where lime had been applied in the field. Even the soil of plots 26, 28 and 31, which had been producing very satisfactory crops as indicated in table 1, did not contain vigorous nitrifying organisms. ‘The rate of nitri- fication was materially increased by applications of calcium carbonate. The nitrifying organisms were much more active in the soil from the manure plots than in the soil from any of the other plots except where lime had been applied. There was a general tendency for the rate of ammonification of casein to decrease with an increase in the lime requirement of thesoils. There weresome marked exceptions to this tendency, notably plots 25 and 26. A study of the analyses of these plots shows a high total content of nitrogen and organic matter. No lime has ever been applied to plots 25 and 26. This increased nitrogen in the form of protein represents an increased amount of material available for the action of ammonifying organisms. If a large amount of nitrogen has been stored up in the soil, the amount of ammonia produced without any applications of calcium carbonate would be sufficient to produce satisfactory yields of those crops which are able to utilize ammonia, on a soil having lime requirements no higher than those of plots 25 and 26. The tendency for small applications of calcium carbonate to be relatively much more effective than larger applications was again shown in these experiments. It was evident that ammonification proceeds fairly satisfactorily without the application of calcium carbonate, especially, as suggested in the preceding discussion, if the content of organic nitrogen is high. Large applications of calcium carbonate had a tendency to reduce the rate of ammonification.
BACTERIAL ACTIVITY AND LIME REQUIREMENT 457
Nitrogen fixation took place very readily in Ashby’s solution when inoculated with soil from any of the plots. There did not seem to beany correlation be- tween the calcium-carbonate requirement and nitrogen fixation. The addi- tion of calcium carbonate to Ashby’s solution caused an increase in nitrogen fixation in every case, but this increase was no more marked in soil having a high lime requirement than in soil having a low lime requirement.
18
4/6
/4
12
Neutral pointeVeitch Method
ee 7 ----<. -
-—_- -——_ —— =
Bacteria in millions per gram of so/l.
oO /000 2000 3000 4000 5000 7500 10,000
Pounds calcium carbonate per 2,000,000 pounds soil. eet SOA With PHOSDUOTUS ~~ ER Soil I with phosphorus. Soil I without phosphorus — - —- — Soil I without phosphorus.
Fic. 1. Toe Errect oF CALcrum CARBONATE ON THE NUMBER OF BACTERIA IN Sorts I AND II
SUMMARY AND CONCLUSIONS
This investigation was undertaken as a preliminary step in the study of the possibilities of a system of acid agriculture on soils somewhat distantly re- moved from a source of lime. A study was made of the relation between the activities of the soil bacteria concerned in nitrogen accumulation and nitrogen transformations and the lime requirement of certain soils. The lime require- ment of these soils varied from none to 4600 pounds of calcium carbonate per 2,000,000 pounds of soil. To different portions of these soils calcium carbon- ate was added in amounts ranging from 0.01 per cent to 2 per cent of the weight
Milligrams nitrogen as ammonia per 00grams soil.
458 FIRMAN E. BEAR
of the soil. The data accumulated show that the various groups of soil organisms vary in their response to applications of calcium carbonate.
Ammonification proceeded fairly satisfactorily in most of the soils without the application of lime. The use of moderate amounts of calcium carbonate increased the rate of ammonification in most cases. Small applications were much more effective, relatively, than large applications.
. — eee ae
vO
60
$0
~
Neutral point Veitch Method ~~
40
ee oO /000 2000 3000 4000 5000 7500 /Q000
Pounds calcium carbonate per 2,000,000 pounds soil. SEY BY Anil Nal ea I Nes Soe Soil I -———- Soi! I with four day period of incubation.
Fic. 2. THE Errect oF CALcrtuM CARBONATE ON THE RATE OF AMMONIFICATION IN Sots I AND II wita PHOSPHORUS
The rate of nitrification was almost directly correlated with the amount of calcium catbonate supplied. Excessive applications were not injurious to the nitrifying organisms. Soils having high lime requirements showed practically no nitrification until calcium carbonate had been mixed with them.
Nitrogen fixation by non-symbiotic soil organisms was considerably in-
———
BACTERIAL ACTIVITY AND LIME REQUIREMENT 459 creased by the addition of calcium carbonate. The application of mono- calcium phosphate also was necessary for maximum nitrogen fixation. All of the soils studied accumulated considerable amounts of nitrogen when in- cubated in Ashby’s solution without the addition of calcium carbonate, although its use increased the rate of nitrogen fixation.
A lime requirement of 3000 pounds was not sufficient to prevent a good growth of soybeans on soil well fertilized with acid phosphate or manure. Nitrogen fixation accompanying the growth of soybeans took place readily
20 —_—
a.
wa anion
~ a
oa
EN
: — — — Soil | with ammonium carbonate. Soil I with ammonium carbonate.
~ is)
WE Ce cea Soil Il with ammonium sulphate. ZO oe s —-—-—Soil I with ammonium sulphate. x ‘ Q 8 ee a ha aS / 3 yr z
Milligrams nitrogen as nitrate per /00 grams soil. %
o) /000 2000 3000 4000 5000 7500 10000 Pounds calcium carbonate per 2,000,000 pourads soil
Fic. 3. Tue Errect of CALCIUM CARBONATE ON THE RATE OF NITRIFICATION IN Sorts I AND II witH PHOSPHORUS
in acid soils. This fixation was increased by small applications but decreased by large applications of calcium carbonate.
From these facts the following conclusions seem justified:
1. Plants which are able to utilize ammonia nitrogen need not suffer from nitrogen hunger when grown on soils having lime requirements no higher than those studied in these investigations. :
2. Plants which depend on nitrates as their source of nitrogen may suffer from the lack of available nitrogen in soils having high lime requirements, unless these requirements have been at least partially satisfied.
460 FIRMAN E. BEAR
3. The supply of nitrogen in acid soils may be maintained by growing acid- resistant legumes, of which the soybean is one. Undoubtedly, the use of acid phosphate aids materially in the nitrogen-fixation processes in acid soils.
4. Small applications of calcium carbonate are, as a rule, relatively more effective than large applications as a means of increasing the bacterial activities in acid soils.
Acknowledgment is due Dr. E. B. Fred of the University of Wisconsin for many helpful suggestions and criticisms offered during the progress of this investigation.
16 14
12
/0
PO ae Oe ee Ss met ae oe ee
Milligrams nitrogen fixed per 100 grams soit.
Neutral point-leitch Method
- = _ -_—
(4) 1000 2000 3000 4000 5000 7500 40000
Pounds calcium carbonate per 2,000,000 pounds of soil With phosphoruS == === - === Without phosphorus.
Fic. 4. Tor Errect or CatciumM CARBONATE ON Non-SYMBIOTIC NITROGEN FIXATION IN Sor II
REFERENCES
(1) Asupy, S. F. 1907 Some observations on the assimilation of atmospheric nitrogen by a free-living soil organism, Azotobacter Chroococcum of Beijerinck. In JOUR PASTAS Cl. V2 Doo.
(2) Bear, F. E., AnD SALTER, R. M. 1916 The residual effects of fertilizers. W. Va. Agr. Exp. Sta. Bul. 160, 25 p.
(3) Berar, F. E., AND SALTER, R. M. 1916 Methods in soil analysis. W. Va. Agr. Exp. Sta. Bul. 159, 24 p., 2 fig.
BACTERIAL ACTIVITY AND LIME REQUIREMENT 461
(4) CHRISTENSEN, H. R., AND Larsen, O. H. 1911 Untersuchung iiber Methoden zur Bestimmung des Kalkbediirfnisses des Bodens. In Centbl Bakt. [etc.], Abt. 2, Bd. 29, p. 347. . (5) Crark, W. M., and Luss, H. A. 1917. The colorimetric determination of hydrogen- ion concentration and its applications in bacteriology. Jn Jour. Bact., v. 2, p. 1-11, 109-136, 191-236. ‘ (6) Covitz, F. V. 1913 The agricultural utilization of acid lands by means of acid tolerant crops. U.S. Dept. Agr. Bul. 6, 13 p. (7) Drew, G. H. 1914 On the precipitation of calcium carbonate in the sea by marine bacteria and on the action of denitrifying bacteria in tropical and temperate seas. In Tortugas Lab., Carnegie Inst., v. 5, p. 9-45. (8) FiscuEer, H. 1909 Ueber den Einfluss des Kalkes auf die Bakterien eines Bodens. In Landw. Vers. Stat., Bd. 70, p. 335. (9) Frear, W. 1915 Sour soils and liming. Penn. Dept. Agr. Bul. 261, 221 p. (10) Frep, E. B., AND GRAUL, E.J. 1916 The gainin nitrogen from the growth of legumes onacid soils. Wis. Agr. Exp. Sta. Research Bul. 39, p. 37. (11) GrmincuaM, C. T. 1909 The formation of calcium carbonate in soil by bacteria. In Jour. Agr. Sci., v. 4, p- 145-150. (12) Hart, A. D., AND MILLER, N.H.J. 1908 Nitrification in acid soils. Jn Jour. Chem. Soc. [London], v. 94, p. 524. (13) Hatt, A. D., AND MILLER, N.H.J. 1911 The production of acids and alkalies in the soil. In Jour. Chem. Soc. [London], v. 100, p. 35. (14) Harter, L.L. 1910 Investigations. Va. Truck Exp. Sta. Bul. 4, p. 80. (15)! Hemricu, R. 1892 Mergel und Mergeln auch die Chemische Bodenanalyzen und ihre Bedeutung fiir die Feststellung der Dungbedurftigkeit der Bodens. In Centbl. Agr. Chem. 1892. (16) HorrMAnn, C. 1906 Relation of soil bacteria to nitrogenous decomposition. Wis. Agr. Exp. Sta. 23rd Ann. Rept., p. 124. (17) HorrMann, C., AND HAMMER, B. W. 1910 Some factors concerned in nitrogen fixa- tion by Azotobacter. Wis. Agr. Exp. Sta. Research Bul. 12; pobt2: (18) Hopkins, C. G. 1910 Soil Fertility and Permanent Agriculture. Ginn and Co., New York, p. 214. (19) Hutcuinson, H. B. 1913. The partial sterilization of the soil by means of caustic lime. Jn Jour. Agr. Sci., v. 5, p- 320-331. (20) Hurcutson, H. B., AND Miter, N.H.J. 1911 The direct assimilation of inorganic and organic forms of nitrogen by higher plants. Jn Centbl. Bakt. (etc.), Abt. 2, Bd. 30, p. 513-547. (21) Ivano, A. 1916 The relation of hydrogen-ion concentration of media to the pro- teolytic activity of Bacillus subtilis. Mass. Agr. Exp. Sta. Bul. 167, p. 141-143. (22) KELLERMAN, K. F., AND ROBINSON, TR. 1910 Legume inoculation and litmus reac- tion of soils.. U.S. Dept. Agr. Bur. Plant Indus. Cir. 71, 11 p. (23) KeLLERMAN, K. F., anp Smrru, N. R. 1914 Bacterial precipitation of calcium car- bonate. In Jour. Wash. Acad. Sci., v. 4, p. 400-402. (24) Kerry, W. P. 1911 The assimilation of nitrogen by rice. Hawaii Agr. Exp. Sta. Bul. 24, p. 5-20. (25) Koretorr, N. 1916 The effect of soil reaction “on ammonification by certain soil fungi. In Soil Sci., v. 1, p. by Ale 4 (26) Kossovitcu, P. S., AND ALTHAUSEN, L. 1907 The influence of calcium carbonate and magnesium carbonate on the soil and plants. In Trudui Mendelyevsk. Syezda Obshch i Prikl. Khim., v. 1, p. 490. Abs. in Exp. Sta: Rec., v.25, (1910), p. 226. (27) Lyon, T. L., AND B1zzELL, J. A. 1913 Some relations of certain higher plants to the formation of nitrates in soils. N. Y. [Cornell] Agr. Exp. Sta. Memoir 1, p. 98.
1 Reference not verified.
462 FIRMAN E. BEAR
(28) Mrtter, F. 1914 Ueber den Einfluss des Kalkes auf die Radenbakeenen In Ztsc Garungsphysiol., Bd. 4, p. 194-206. ; i,
(29) Scares, F. M. 1915 Relation of lime to production of nitrates and mineral nitrog Abs. in Science, n. s., v. 42, p. 317.
(30) Suarp, L. T., anp Hoactanp, D.R. 1916 Acidity and adsorption in boils as AP yf by he hydrogen electrode. Jn Jour. Agr. Research, v. 7, p. 123-128.
(31) Tempte, J. G. 1914 Nitrification in acid or non-basic soils. Ga. Agr. ae Bul. 103, p. 14-15.
(32) Truoc, E. 1916(a) A new apparatus for the determination of soil catonies
new methods for the determination of soil acidity. In Jour. Indus. Engin. Ch
v. 8, p. 345-348.
(33) Truoc, E. 1916(b) The cause and nature of soil acidity with special regard to colloids and adsorption. Jn Jour. Phys. Chem., v. 20, p. 457-484. ih,
(34) VerrcH, F. P. 1904 Comparison of methods of estimation of soil acidity. In ‘Tous We Amer. Chem. Soc., v. 26, p. 661. f
(35) VoorHeEss, E. B., anD ae J. G. 1907 Some chad and bacterial effects of — liming. N. J. Agr. Exp. Sta. Bul. 210, p. 178. °
(36) WHEELER, H. J. 1897 The fifth year’s observation upon the growth of plants upon an acid upland soil, limed and unlimed. R. I. Agr. Exp. Sta. Rpt., 1897, p. 224-225.
(37) WHEELER, H. J. 1903 A further study of the influence of lime upon plant growth. R. I. Agr. Exp. Sta. Bul. 96, 44 p.
(38) WHEELER, H. J. 1914 The comparative effect on different kinds of plants of liming on acid soil. R.I. Agr. Exp. Sta. Bul. 160, p. 407-446.
(39) Wurttnc, A. L. 1913 Soybean and cowpea nodules. In Breeder’s Gaz., v. 64, p. 1130.
(40) Wurtson, A. R., AnD Weir, W. W. 1913 Soil acidity and liming. Wis. Agr. Exp. Sta. Bul. 230, p. 1.
(41) Witurams, C.G. 1916 The effect of lime on acid soils. Ohio. Agr. Exp. Sta. (Un- published data).
hi : Has
5