ra G4% “13 4 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