iri LIBRARY Michigan State University eee PLACE IN RETURN BOX to remove thie checkout from your record. TO AVOID FINES retum on or before date due. MSU Is An Affirmative Action/E qual Opportunity Inetitution c\ckre\detedus.pm3-p. 1 THE BeBECT OF SOMK ACIDS, ALKALIS AND INORGANIC SALTS ON SOIL BACTERIA Ii] THis SOIL SOLUTION. THSSIS Submitted to the faculty of the Michigan Agricultural College in partial fulfillment of the requirements for the degree of Master of Science. Oswald Martin Gruzit, B. S. June 1916. JHES'¢ The Effect of Some Acids, Alkalis and Inorgsenic Salts on Soil Bacteria in the Soil Solution. 1. Introduction. ze Consideration of the sources of error. 5. Historical review. | A. The effect of acid and alkalin conditions of the medium on microorganisms. B. The effect of reaction in the soil on soil bacteria. a. The effect of reaction on nitrifying processes in soil. 0. The effect of reaction on denitrifying processes in soil. ec. The effect of reaction on ammonifying processes in soil. ad. The effect of reaction on symbiotic and nonsymbiotic processes of nitrogen fixing in soil. 4. Method of investigation. 5. Experimental work. A. The effect of the reaction on soil bacteria in the soil solution. a. The effect of hydrochloric acid. b. The effect of sulphuric acid. B. The effect of some inorganic salts on soil bacteria in the soil solution. 6. General discussion. 7. Summary. 8. Acknowledgement. 9. Reference list. oe nay O wo The Effect of Some Acids, Alkalis and Inorganic Salts on Soil Bacteria in the Soil Solution. Introduction. The effect of acids, alkalis and inorganic salts on the microflors of the soil is a subject of immense practical and theoretical value for the explanation of the complex prob- lems of the soil fertility. The majority of the common crops(1) fail to grow on acid or moderately strong alkalin soils. The significant rdle which the microorganisms play in the nutritim of higher plants has become . common knowledge. It is generally considered that the development of bacteria does not proceed at the best on a soil with unfavorable reaction. The interdepend- ence of crop growth, unfavorable reaction of the soil and the reduced bacterial activity in such soils lead to the exhaustion of the soluble mineral elements necessary for best development of plants(2), Little or no attention has been directed to the study of changes in the bacterial flora of the soil solution at different acid and alkalin conditions. Such study would aid materially to ascertain the resisting power of soil bacteria to the unfavorable conditions of reaction and would lead to the knowledge of the limits of reaction between which the soil bacteria are most active. Consideration of the Sources of Error. The complexity of the soil mass with its manifold and constantly changing processes presents many objectionable fea- tures for the study of the effect of reaction and salts on the -2-. soil bacteria due to the chemical interaction occurring petween the added compounds and those that were present in the soil. Loeb$3), Osterhout(4) and others(5) have added knowledge tmthe effect of the antagonistic ection of various bases. The exchange of bases(6), the effect of colloids vs. crystalloids(7) and the double salt formetion(8) which probably occurs in the soil introduce many unforeseen features which may lead to entirely unexpected results when the soil itself is used for the study of the effect of chemicals on the soil bacteria. The coordination of the solid phase with the liquid phase of the soil, tne difficulties experienced in chemical analysis of the two phases when together in soil, the errors which enter in determination of the reaction of the soil by the present methods(9), do not offer inducement for a quenti- tative study of the effect of vsrious chemicsl on soil bacteria. In view of this the liquid phase of the soil was extracted from the soil and used for the audy of the effect of reaction and chemicals on tne soil bacteria. The objections to the use of the soil solution instead of the soil itself are of, nature not fully xnown. To what extent tnis soil solution is so true and unaltered soil solution can not be stated definitely at present. Istecherikow(10) thinks that the soil solution ex- tracted by the alcohol displacement method has the same chemical composition as the soil, from which it was extracted. The un- published data on the oil pressure method(11) give strong assur- ence that prectically no change in concentration of the soil Solution occurs in process of the extraction. The microflora of the soil solution when extracted by the oil pressure method u is lower when compared with the soil itself. In Table I,is seen - 3- that the number of becteria is several times less in the soil solution than in the sofl itself. However, it is natural to expect a lower number of bacteria in the solution than in soil, Table I. Sandy Soil Sandy Loem Soil (one gram oven dry soil)* 3,424,000 9,835 ,000 Soil solution (one c.c.) 1,150,000 1,429,000 because the extraction of the soil solution is worked on the principle of the filtration plants for purification of water. However, tue types of bacteria did not appear different which must be expected because the soil from the lower strata under- goes little or no filtration, the organisms are carried out by the flowing soil solution. The soil solution undoubtedly provides modified conditions because it is separated from the solid phase, yet for the study of the effect of acids, alkalis and salts on soil bacteria it possesses merit worthy of con- sideration. *Counts in soil and quartz sand were always made on oven dry bases (24 hrs. at 105°C.). ~4- Historical Review. Activity in the investigation of the causes of acid or alkalin reaction in the soil and their effect on living organisms has been extensive during the past years. In main the investigations bear out the idea that the toxicidy in the soil might be due to one or several of the following four causes: Presence of a free organic acid; adsorption of bases, With consequent lineration of acid radical; presence of free inorganic acids;and due to toxic substances of organic or in- organic nature. Kossowitch(12) supposes that the reaction in soil results from the geological composition of the soil. While the reaction is an unavoidable result due to mineral composi- tion, it dominates the chemical and biological activities of the soil. Gedroitz(13) assimes that acid or alkalin condition of the soil solution is due to the presence of free hydrogen and hydroxyl ions. Harris(14) presents expdrimental facts to show that acid condition in so0il arises through adsorption of bases. Salts, of aluminum nitrated have been found by Abbout, Conner and Malley(15) to be very toxic to plants. Brown, Howe and Sar(16), summarizing the causes for acid production in the s0il state tnat the decay of organic matter, supplied in a form of manures, cover crops and crop residues, leads to a large production of various organic acids. Mineral fertilizers, like (WH, )gS504 or KCl when added to the soil give rise to sulphuric and hydrochloric acids. In the process of nitrification nitric acid is produced. The Effect of Acid and Alkalin Condition of the Medium Nicroorganisms. It is general knowledge that microbial activities are retarded in the presence of unsuitable reaction of the medium. The effect of acid or alkalin reaction varies with the kind of organisms. iiolds prefer slightly acid reaction. Bacteria vary in their requirements of reaction. Some are not impaired by fairly strong acid reaction, some prefer Slightly alkalin condition and are killed by acid reaction. Rahn(17) has shown that the lactic acid bacteria in milk develop rapidly up to about 0.3% acid. Soon after the reac- tion of milk reaches 0.816% acid the bacterial life is supressed by their own by-products. To quote Esten and Heason(18): “On souring of milk many of the several varieties of bacteria commence to increase along with the lactic acid bacteria, but the miscellaneous varieties are soon checked by the acid produced by the lactic acid bacteria as they are nore sensitive to acid than are the lactic acid bacteria. By this time milk curdles from the acid produced, most of the miscellaneous bacteria are killed or pickled". The fermenta- tion of sauer kraut according to Webmer, Konrad and Lehmann(19) is analogous to milk fermentation: "The acid bacteria grew until they produced sufficient acid to inhibit their own srowth, after which they gradually died out". In fermenta- tion of apple juice according to Esten and Mason(19) acteris fail to develop until the acidity, wich is found to be 0.72 percent in terms of normal NaOH, is net neutralized. In the study of the fermentation processes of silage, Esten and Mason(19) have shown that there is a marked decrease of number of bacteria with increase of acidity in the silage and that eimest-norre but a few acid bacteria survive when the reaction reaches 1.3 to 1.86 percent. acid of mixed lactic and acetic acids. the Effect of Reaction in the Soil on soil Bacteria. The relation of soil bacteria towards the acid or alkalin reaction has been under prolonged consideration, yet 4 nec no definite results are drewras to the exact limitations of tolerance of microfigganisms towards reaction. Fabricus and Feilitzen(20) in their study of the bacteriological content of Hochmoor, peat soils, have found that these soils are very poor in bacteria which is probably associated with the high acid reaction of these soils. Through cultivation and liming these soils developed very large numbers of microorganisms. Lipnan(21) says: “Increasing acidity will retard more and more the fermentation changes in the soil, and the bacteria themselves will dwindle in numbers. Finally the soil may become unfit for the natural growth of higher and lower plants and will cease to yield profitable harvests". On examining the literature concerning the specific groups of soil bacteria, as free nitrogen fixing, nitrifying denitrifying, ammonifying, and sulphofying bacteria as to their tolerance of acid and alkalin reaction, one finds only general statements. Hall, Miller, and Giningham(22) have shown that in acid soils the process of nitrification goes on very slowly, and that the number of nitrifying bacteria is very low. However, Temple(23) shows that nitrification goes on in presence of organic acids, but the rate of nitrification is less in poor soils than in rich. Allen and Bonazzi(24) express the accepted view by stating that application of lime stone imparts vigorous nitrifying power to acid soils. Lipman and Burgess(25) show further that 0.45 percent of total alkali may be present in Boil to permit practically normal nitrifying processes. The process of denitrification by bacteria is generally — - 7 e thought to proceed more rapidly in soils with wet unaltered conditions. Such soils often are slightly acid. Klaeser(26) in his study of the effect of the reaction on reduction of nitrates found that this process is controlled by three factors; namely, the specific organism, the reaction of the medium, and the specific nitrate compound used for the study of the reduction processes. When the nitrate salts of potas- sium, sodium and calcium were used in presence of one pércent dextrose, B. megatherium did not attack any of the salts, while B. oxalaticus grew best in acid conditions, deoxidising only the potassium and sodium salts. 8B. subtilis, on the con- trary, grew best in a neutral medium and deoxidised all three salts first to nitrites and later to ammonia. B. tumescens produced the best growth in acid medium and deoxidised calcium nitrate with less rapidity than the potassium and sodium salts changing the salts directly into ammonia. The process of ammonification according to Lipmar?27 ) is seriously impaired by small quantities of alkalin salts such as sodium chlorid and sodium sulphate. Contrary to this, sodiun carbonate has a stimulating effect when present in quantities not exceeding 0.10 percent. of the dry weight of the soil. Acid condition in the soil retards amnonification, yet Temple's(23) view is that the presence of some acid radical may exert beneficial action on bacterial processes on account of neutralizing the free ammonia. The non-symbiotic free nitrogen-fixing bacteria, namely, the Azotobacter group, are invariably depressed in their activity in absence of lime and other basic substances. Fisher (28) summarizes the previous researches and advances facts to show that the minimum lime (CaO) content of the soil must - 8 « approach 9.10 percent to sustain the life processes of the organisms. There is sufficient experimental and practical evidence to show that most of the symbiotic nitrogen-fixing bacteria associated with the legume plants require an alkalin condition of the medium and, with few exceptions, are entirely sSupressed in acid media. The most suitable reaction according to Harrison and Barlow(29) for the Ps. radicicola sp. is in the neighborhood of neutral to -3°. However, the variety of Ps. radicicola associated with lupine and serradella plants can not tolerate alkalin reaction of the medium. Chandurin(30) shows that yellow lupine grown in rich soil with alkalin re- action will not develop nodules on the roots. When transferred to acid soil the nodules develop readily. From the above review of the literature it is ine- ferred that the sensitiveness of bacteria to the reaction of the medium is quite noticeable. The important groups of soil bacteria prefer alkalin reaction of the medium. There is little information concerning the limits of reaction tolerated by the various groups of soil bacteria and the specific reac- tion which is most suitable for the development of soil bacteria. Method of Investigation. The soil solutions were extracted by the oil pres- sure method(1z) from rich sandy and sandy loam soils and ad- justed to various degrees of reaction with N/100 mineral acid and N/100 iwaOH, using methyl red as an indicator for titration of the soil solution. The chemical composition of the soil Solution was changed by the addition of potassium nitrate and phosphate salts. One hundred grams of a pure medium coarse sterile quartz sand, prepared by treating with strong aqu-a -9 « regia (Hii0,:HCL=1:2) for two hours and washing with distilled water until free from chlorides and nitrates and then ignited to free from traces of ammonia, were mixed thoroughly with 9 c.c. of the adjusted soil solution in case of the study of the effect of chemicals on bacteria and with 10 c.c. for the study of the effect of the reaction. The sand cultures were kept in 200 c.c. resistante glass sterile erlemeyer flasks plugged with cotton and stored in the dark at temperature varying between 19° and 23°C. The moisture content of the cultures was adjusted with ammonia free sterile distilled water about every ten days. Bacteriological counts were made on Conn's(31) sodium asparD- ginate agur after 7 days' incubation period at 19°-23°C. fFor making dilutions of sand cultures to the totad amount pf sand was added enough of sierile ammonia free distilled water to make the moisture content 100 percent, thus making dilution 1:1. The contents were well shaken and higher dilutions made up to 1: 100,000. The two highest dilutions were used for plating. Du- plicate plates were always made from the highest dilution. For every plating duplicate cultures were used. By using the whole sand culture to make the initial dilution 1:1 eliminated the laborious process of weighing one gram portion and undoubtedly reducing the chance for contamination add minimizing the error for securing a representative sample. The reaction of the sand culture was determined by taking a portion of the liquid from the 1:1 dilution, filtering and testing the filtrate with methyl red. At the last plating of the last cultures used for the study of the effect of reac- tion on bacteris, the filtrate was titrated with n/100 Hz50,4 acid and n/1000 NaOH to sscertain the change of reaction in the Cultures quantitatively. -10- Bacteria from the cultures with acid reaction were isolated and studied es to their morphological and physiological characters. The physiological character of the organisms was studied to determine their nitrifying, emmonifying and denitrifying power in the synthetic media. ~-ll- Experimental Work The study of the microflors of the soil solution was undertaken with the particular aim to determine the resisting power of soil bacteria to unfavorable reaction of the soil sobu- tion and to advance, if possible, some suggestion which would lead to better understanding of the soil fertility problems in the acid soils. ‘io secure more complete data as to changes oc- curring in the sdl solution the latter was also studied from the standpoint of chemical composition. Both the reaction omd chemical composition of the soil solution are undoubtedly de- ciding factors in the activities of soil bacteria. So far as can be discovered from the literature, there is no complete explanation as to the effect of the reaction snd chem- ical composition on the soil bacteria. This undoubtedly is due to our incomplete knowledge of the chemical, physical, asd bio- logicel processes occurring in the soil which lead to the change in the composition and reaction. From the review of the previ- ous investigations, it is seen that the reaction and the chemi- cal composition of the soil exert a strong influence on the soil bacteria. It apped s of importance to emphasize the independeace of the reaction and the composition of the soil solution not only as factors’ influencing the biological phase of the soil solution, oat also as suggesting the chemical changes which occur in the soil and thru such changes the effect of the biological factors on the soil. With this in view, the experiments were arranged in two groups: (1) The effect of the reaction on soil bacteria in the Bpil edlution, and (2) The effect of some inorganic sakes on soil bacteria in the soil solution. -12- The £2¥fect of the Reection on Soil Bacteria in the Boil Solution. To demonstrafe the effect of an acid reaction on the soil bacteria hydrochloric and sulphuric acids were used to sé- juetré¢he reaction of the soil solution. The Effect of Hydrochloric Acid on Soil Bacteria The soil solution was extracted from a Miami sandy soil on wnich alfalfa was growing. The extracted soil solution took 2.24 cc. N/100 HCl per 10 c.c. to neutralize, ot it required 2.24 c.c. N/l HCl to neutralize one liter of the solution, whict: is equivalent to N/446.4 alkali in terms of NaOH. It requires 0224 c.c. of N/100 HCl to neutralize 1 c.c. of the soil solution. To raise the reaction to 1/1200 it required 0.0833 c.c. To neutralize and raise the reaction to N/1200 acid takes .30733 c.a of N/100 acid per 1 cee. of the solution. To every resistance glass erlemeyer flask containing 100 gms. of oven dry pure sterile quatts. sand there were added 10 c.c of the adjusted soil solution . For comparison of the effect of hydrochloric acid on soil bacteria, parallel flasks with unaltered end neutral soil solu- tions were carried exactly as those with acid reaction. ‘There were 16 flasks for every reaction. For control check flasks con- taining qmartz sand and 10 c.c. distilled water were carried. The experiment lasted about eight months from October 7, 1915 to May 20, 1916. In Table I sre shown the changes in the number of bsecteris. in the soil solution under different reactions of the medium. -13- Table I The Effect of Reaction on the Number of Soil Bacteria Days Beginning 18 36 60 117 132 146 207 soil 3, 424% Soil Solution unaltered 1,393 1,415 2040 1150 465 612 485 272 alk. N/446.4 : , Neutral N/1lO_ " 1,320 1,005 1,040 792 317 410 55 Acid HCl N/1200 * 26 28 8.7 44 12 27 8 * The number of bacteria expressed in thousands per gram on oven dry bases of sand. The changes that occur in the alkalin and the neutral solutions are gradual, but persistent. ‘The reaction of N/1200 acid is very toxic to bacteria. After the eighteenth day of incubation the number of bacteria in these Cultures was reduced by 98.9 percent when the number of bacteria in N/446.6 alkalin solution was taken for comparison, thus s&owing that only 1.10 percent of bacteria can survive N/1200 acid reaction. The study of the colonies on eddiam asparaginate agar plates ald in pure cultures of the isolated organisms revealed that es far as the colonies appear on plates they were similer, however, in studying the isolated organisms it was shown that they were not the same organisms. During the eight months period there were isolated eight different types. Some of these or- ganisms were spore-bearing, some were not. Their cultures end physiological characters will be described un the the study of the morphological and physiological character of the isolated orgenisms. The types of bacterial colonies from cultures with alke- lin and neutral reactions were numerous, but like in appearance With the age of these cultures the different types of the colo- nies were gradually reduced to few. With the increase of the age of the cultures the reaction of the cultures underwent gradual change. Table II shows that there was a perceptible change of reaction at the thirty-eighth day. It took about fifty-six days to neutralize the alkalin reaction and produce enough of acid to be demonstrable when amalysis was made at the sixtieth day. Table II. Days Beginning 18 356 60 117 132 146 207 Unaltered Soil solution Alk. N/446.4 Alk. Alk. Alk. Acid Acid Aéétd Acid Acid Neutral Neut. Acid Acid Acid Acid Acid Acid Acid Acid N/1200 HCl Acid Acid Agid Acid Acid Acid Acid Acid The cultures in which the reaction was akjusted to neutral con- dition indicated slight acid reaction after 18 days of incuba- tion. The number of bacteria in these cultures never showed an increase, but was always on, decline, and when the reaction be- ceme acid, the decresse was rapid. The curve of the culture with neutral reaction, as shown in Figure 1, in a striking degree approsches a parallel direction to the curve of the culture with malkalin reaction after the thirty-sixth day of the experiment. The curves in Figure 1 essentially suow the changes of the number of bacteria with the changes of the reaction of the cultures. - 15 «- The culture with acid reaction at the start of the experiment shows an abrupt drop in the number of bacteria, wnich never shows the NenlendTo a GraduroR Anrentcd, that, bacteria would,be on the incresse, but fluctuate within narrow limits. ‘The reaction of the original neutral and N/1200 HCl acid cultures titrated after 207 days as follows: Original sdl solution +.044°F.5S. Neutral +.Q018°F.S. N/1200 HCl Acid +.166°F.S. As a resume of this experiment interesting segestions can be made. First, under the conditions of our experiment the cultures with alkalin and neutral reaction undergo gradual change of reaction from akkalin to neutral, ad from neutral to acid condition. Second, with the change of the reaction there occurs gradual increase of bacteria up to the point where the reaction is barely alkalin, Third, with the change of the reatét¢tion from barely alkaline to neutral- and acid reaction, the number of bacteria gradually decreases. Fourth, there is reason to sup- pose that at no time 98.8 percent of the soil bacteria from a sandy soil can endure acid reaction of N/1200 acid (+0.83°F.S.). = [b= MECHANICAL ENGINEERING DEPARTMENT M.A. C. Fieure |. - 17 -~ The Iiffect of Sulphuric Acid on Soil Bacteria in the Soil Solution For the study of the effect of sulphuric acid, the soil solution was extracted from a rich orchard Miami sandy loom. The reaction was adjusted and retitrated once more showed the following strengths: Unaltered soil solution. Alkalin 4/1040 i i " Made alkalin W/990 “ i" i" " " N/412 " " " Ne utr al Neutral " " " Acid N/1200 i i" fn " N/1400 " ft ‘ Hl N/2164 " 8 i " N/2840 To neutralize 100 c.c. of the soil solution it required 9.61 c.c. of N/100 HgSQ, acid. According to the following formu- la, 10,000 + 9.61* the strength of the soil solution is equiva- lent to N/1040 eadkalin. This formula of course is simplified expression of several steps of proportions for calculating nor® medity of a solution. Ten cubic centimeters of the adjusted soil solution were mixed thoroughay with 100 grams of sterile pure qmartz sand and incubated at the same conditions as the culture flasks for the study of the effect of hydrochloric acid on soil becteria. The experiment was begum November 28, 1915 and lasted until February 13, 1916. * 9.61 - number of cc. of N/100 acid to neutralize 100 c.c. of the sélution. - 18 e- The moisture content of the cultures fluctusted con- siderably yet the bacterial count does not indicate that de- crease or increase of the number of bacteria fluctuated accord- ingly. From Table V it is seen that the greatest difference in mean vatiation is 0.66 percent. Table V. Fluctuation of Moisture Content Days 6 LO 16 67 83 lMeange:- for all perjod Unaltered soil solution akk. N/1040:9.3% 8.2% 8.0% 8.3% 8.5% 8.46% Alkalin N/990 8.8 8.2 7.6 8.0 --- 8.15 Alkalin N/412 9.1 8.0 7.7 7.9 17.7 8.08 Neutral w/O 8.9 8.5 --- 8.4 17.9 8.30 Acid Ha2S0, N/1200 9.0 8.2 7.4 8.4 8.2 8.24 Acid " N/1400 9.2 8.5 7.6 8.3 8.2 8.32 Acid " N/2164 7.9 8.2 7.2 8.0 17.7 7.80 Acid "* N/2840 8.0 7.7 7.5 8.2 7.6 7.80 Average of moisture 8.8 8.2 7.6 8.2 8.0 The data in the Table VI..addthe curves in Figure 2, representing the changes in number of bacteria in the sand Cultures adjusted to various reactions in the main bring out the facts: First, that the cultures with alkalin reaction whow great increase of number of bacteria in the first ten to sixteen days after which time the growth of bacteria decreases rapidly. Second, the cultures with neutral reaction support very feeble development of bacteria. Third, The cultures with acid reaction, namely with the reaction below N/2164 show no or very slight Tesistance to the acid reaction. Table VI The Effect of Sulphuric Acid on Soil Bacteria in the Soil Solution — - Days Begini ning 6 10 16 67 83 Soil 9335* Soil solution une aktered akk.N/1040 1329 4200 5850 3700 9465 830 Soil solution made alk. N/9$0@ 4000 5000 5780 1505 --- " " 4/412 2625 5927 6605 1350 # £830 " Neut N/O 2386 2472 <-e-- 1197 1070 " Acid N/1200 " " 4/1400 " " M/2164 " " N/2840 18 335 22 479 482 985 140 220 1470 1102 1042 1450 597 1822 2980 3855 905 aon a 7 el eet Z22z22es228 wo : a © * The number of bacteria expressed in thousands per gram on oven dry basis of sand. The cultures with the reaction of 1/2840 at the be- ginning showed to be better medium for the development of bac- teria than the cultures with the neutral reaction. However, after sixteen days the number of bacteria decrease rapidly. The bacteria in the cultures with alkalin media develop most rapidly in the case where the reaction is lowest (N/1040 alkalin). Wext in order of rapid development are cultures with W/990 alkalin reaction. ‘The climax of development in cultures with N/412 alkalin reaction is reached only after sinteen days, thus showing that the N/4l1l2 alkalin reaction at the beginning had a retarding effect on the bacterial growth, butt was partly neutralized by the bacter@al processes, which appear as shown by the number of bacteria, at about the eighth day of in- cubation, the growth was rapid. When the number and the rapidity of development of bacteria in these cultures and that in case of cultures with neutral reaction are compared it is strongly sag- gestive that the most suitable reaction for the development of -20- soil bacteria is about N/1000 alkalin (-1.0°F.S.). It is in- teresting to note the sensitiveness of the soil bacteria to the reaction. The difference in reaction between N/1040 amd N/990 is only (1.01° - 0.96°) -.05° F.S. and yet it shows the effect until the cultures become acid in reaction. the growth of soil bacteria in cultures with acid reaction due to HgSQ, shows the same gemeral tendency as in the case of hydrochloric acid. The N/1200 (+.83°F.S.) is the crucial point for the soil bacteria. In this case only 0.6 percent of bacteria can withstand so high H-eion concentration. With the lowering of the acidity in the cultures, the number of bacteria does not undergo such abrupt destruction as in cultures with N/1200 acid reacti@n. Contrary to this, when the reaction is as low as N/2800 H2,80,4 acid, there is slight multiplication of bacteria. However, they ave not capable of multiplying half as rapidly as in the case of bacteria in cul# tures with N/1040 alkalin reaction. Yet they multiply slowly up to the sixty-seventh day when there is general decrease of number of bacteria. The results in Table VI indicate that the decrease in number of bacteris is associated with the change 6f the reaction in the culture media. The curves in Figure 2 vring out this point clearly. ~41- MECHANICAL ENGINEERING DEPARTMENT M.A. C. Ficure 2. Unfortunately there were no counts made after the six- teenth day of incubation up to the 67th day. On account of this it is impossible to say whether there would be any further increase in the number of bacteria in the cultures with N/990 ead N/412 alkalin reactions and thus to present some knowledge as to the specific degree of alkalinity at which the majority of the soil organisms thrive best. In further studying the number of bacteria in various cultures and the corresponding curves in Figure 2, an interest- ing fact is brought out. the increase @& number of bacteria in N/1040 add N/990 is pmrallel and continuous. However, after the tenth day the number of bacteria in N/LO4O alkalin culture drops down very rapidly, watle the number in N/990 alkalin culture increases em@d reaches practically the same number and drops down after the sixteenth day. If, in this connéction the curve of N/412 alkalin solution is,studied, it is seew that the increase in number of bacteria in this culture in six days is only half as rapid. However, after the sixth day the increase is rapid and continuous above the number in N/1040 and N/990 alkalin reaction cultures. To explain the unequal growth of soil bacteria in the different alkalin curves, it appears that there is a definite reaction which is most favorabie for the bacterial development. According to the data here presented this reaction appears to be in the neighborhood of N/1000 alkalin. When the reaction is N/990 alkalin, the growth of bacteria is slightly inhibited, but in six days it undergoes Slight neutralization add conseqently the number of bacteria continues to develop at the end of the sixteenth day, while in the cultures with N/1040 alkalin reaction, the solution had - 23 - passed the neutral point at the tenth day. In the N/412 alkalin reaction cultures the reaction was too great to produce apprecia- ble growth of bacteria at the end of the sixth day. However, after the sixth day the reaction became less strong and we see an enormous increase of bacteria which is continuous up to the sixteenth day and probably would continue a few days longer until the reaction would become acid. The changes in reaction of the cultures are given in Table VII. At the sixteenth day the reaction in N/1040 akkalin solution was slightly acid, while in the N/990 alkalin it was still slightly alkalin. The N/412 alkalin culture had"alkalin reaction. The growth of soil bscteria in cultures with acid reac- tion is probably what could be expected. With the highest hydrogen ion concentration the number of bacteria decreased to an average of 31,000 per c.c. and were fluctuating around thgs number. In the cultures with less H-ion concentration (¥/1400 acid), the germicidal action is not as marked.as in the case of Table VII. The changes in reaction of the sand cultures adjusted with sulphuric acid Days Beginning 6 10 16 67 83 Soil solution un- Sl. altered alk. N/1040 Alk Alk. Alk. adeid acid acid " made alk. N/990 Alk Alk. Alk. S.Al. Acid Acid n wW/412 Ali Mik. Alk. Alk. Acid Acid a Neutra ut. Neut Neut ---- Acid Acid " Acid N 1200 ota Acid Acid Acid Acid Acid " " N/1400 “ " " " " " #8 t N/2166 " * #0 " " ¢ “4 r N/2840 " ‘ " " 5 1 N/1200 acid cultures. In these cultures the number of bacteria - 24 « decreases not as rapidly and not to such a low number in the First six days, even after the tenth day they are pultiplying. However, after the sixteenth day the multiplication is entirely inhibited, and they. gradually decrease in number. With the reaction of N/2164 acid the processes of bacterial nultiplicas¢ tion on.4 whole are at zero. During the first six days they undergo Slight develppment following witharather abrupt drop between sixth and sixteenth days. From here on there is Slight multiplication of bacteria, but the process is not intensive enough to rise above the highest number at the sixth day. In the cultures with the reaction of N/2840 acid, the multiplication of bacteria is not suppressed. The number of bacteria are on increase up to t:e sixteenth day after which there is a gradual decline iff number of bacteria. The culture With neutral reaction shows an increase in number of bacteria in aperiod of ten days, after which time plating was not made until the 67th day. On account of this, it is impossible to judge how long the cultures would allow multiplication of the orgenisms. At the end of the sixty-seventh day the number of bacteria in all cultures, with the exception of higHest hydrogen ion concentration, reachmpractically the same standard. From the foregoing data the following suggestions might be advanced: First, the growth of soil bacteria, when Studied in the soil solution of sandcultures, is stimulated when the reaction of the medium is approximately N/1000 (-1.0°F.S.) alkalin. Second, the cultures having the medium about W/2200 - 25 - (+0.45°F.S. jrofahigher hydrogen ion conceniration, are to 4 certain degree germicidal to the majority of the soil bacteria. The cultures with lower hydevgen ion concentration stimulate slight developmant of bacteria for a short period and then the reaction becomes inhibitive to the growth to the greatest number of soil bacteria. Third, the reaction in the alkalin cultures gradually is neutralized end later becomes acid in reaction. The Effect of Some Inorganic Salts on Soil Bacteria in the Soil Solution. Before Loeb's (3) work on the role of balanced solutions on the life processes of lower organisms, application of Zerti- lizers and other salts to the soil was considered to have only nutritive value to the plants. It is known that application of sodium chlorid. to some soils may stimulate the crop producing power, yes this salt is not recognized of having nutfitive value for higher plants. the addition to the soil of small quantities of commercial fertilizers result in increased plant growth which in some cases is too great to account for the amount of the fer- tilizer used. Can it not be supposed that addition of fertili- zers to the soil stimulates the bacterial activities which lead to increase of soluble food for plant growth? Recent work of Fred and Hert (32) emphasizes distinctly the important réle of various sulphate and phosphate salts on the bacterialactivities in the soil. In the study of some alkalin salts as affecting soil bacteria, Lipman (33) has found that bacterial activities, hamely, nitrification process, are inhibitzde when the soil contains my of the following salts in, amounts cquai—te 0.025 percent, sodium cabbonate, 0.35 percent sodium sulphate, and - 26 - O.1 percent or less of sodium chlorid. licLean and Wilson (34) state that acid mmikgnux phosphate when used in large amounts caused a decrease in the number of bacteria. Lipman (35) | reports that calcium sulphate has a veneficial effect on the development of bacteria. According to Stocklasa (36) and Fedd and Hert (32) phosphate salts of potassium show a distinct veneficial effect upon bacterial development. The sulphates 6f potassium and calciim have apparently no effect on total number of soil bacteria (32). This is also confirmed by Pitz (37). The role of the various salts in soil is far from known. It appears that their efrect on bacterial development and the changes produced in the reaction of the medium duringa prolonged period has not been studied. It is the purpose of this work to study the effect of some inorganic salts upon soil bacteria and to imdicate changes in the reactien of the medium. Standard solutions of nitrogen, potassium and phosphorus were prepared from pure salts containing one or more of these three elements in such concentrations that when one c.c. of the standard is added to 100 grams of sand, the mixture will contain, in parts per million, 33, 30, and 23.75 paets of nitrogen, potassium, or phosphorus respectively. The standard solutions are as follows: KCl 0.57164 KH3P0«4 | 1 .0433% NaNOg 2.0000% NagHP0O,4 .12H30 2.7421% One hundred grams of dry pure quartz sand were weighed out and placed in resistant glass flasks to which the - 27 - standagd solutions were added according to the following outline: No. of flask Standard solution lwo. of cc. per 100 gms. sand Ree 1 = 16 KCl 1 cc. 17 = 32 NaNQs l cc. 33 - 48 NagHPO, lcc. 49 - 64 KCl + NaNO, 1 cc. +l ce. 65 - 80 KHgP04 l cc. 81 - 96 NaNOg + NagHPO, lcec. t+ l cc. 97 - 112 KHaP0, + NaNOs l cc. + l ce. 113 - 128 check rr The flasks containing the sand and the standard solutions were dried amid sterilized in a hot air oven. After sterilization 9 cc. of the soil solution which according the official methods of soil analysis contained, in parts per million, 50.7, 10.8, and 34,.8 of nitrogen, potassium, and P30, respectively, were added to make the moisture content 9 percent ion the basis of oven dry sand. At the same time were carried check flasks one set of which contained quartz. sand and soil solution alone and xtmm to’ the other set, instead of soil solution, was added ammonia free distilled water. The number of bacteriacapable of making a perceptible growth on sodiun asparaginate agar were determined at irrggular intervals and tabulated as the number per gram of oven dry sand. The moisture content of the cultures was adjusted at the same time as in case of the study of the reactions and consequently fluctuated in the same limits as shown in Table III. Percent of moisture at the time of plating is given in Table IX. - 28 « From the dataitis seen that the moisture fluctuation had no effect on the chamges in number of bacteria. The results in Table VIII show the effect of the salts and the effect of the duration of the experiment on soil bacteriate Addition of the single salts of potassium chloride, of dibasic sodiwn phosphate and of sodium nitrate stimulates the develop- ment of microorganisms in comparison with the check to which no Salt was added. Combination of salts in pairs, such as KCl + NaNOy, and NagHPO, + NalNOg have the same initial effect upon soil bacteria as the above salts. whe monobasic potassium phosphate does not show any effect upon the Boil bacteria Within sixty days. The number fluctuates within narrow limits of the count in the original soil solution. The three nutrient @lement combination of monopotassium phosphate and sodium ni- trate show the largest increase of number of bacteria on the eighteenth day. When the effect of salts upon the continued high numbers of microorganisms is compared, itcis found that different salts have a variable effect, Potassium chlorid in thirty-six days has produced a distinct inhibitive condition for the growth of bacteria. Sodium nitrate, dibasic sodium phosphate and combins- tion of potassium chlorid plus sodium nitrate produce similar conditions in the medium between thirty-six and sixty days .The two Sait sombination of sodium nitrate and dibasic sodium phosphate andacombination of monopotassium phosphate and sodium nitrate became toxic to bacterial flora between 117 and 132 days. Mono- potassium phosphate shows a toxic effeat between 60atd 117 days. Acoarding tu:the initial eftect on the development of bacteria, the above data may be classed into four groups in order of the n Millions. B » ~ 12 Lo 04 0-b oy iB 20 Fieu rs 2. The ~% Heel of Snarqanic Salts on Number of: Soil acterca on the Soil Solution. - Re ~ MaNOs + Na, HPO, a ot Diem ore nib Tika e oe Ja x ‘i Ovigi nal Soul SOhition eg Zee - = $ ott - 29 «- Table VIII. Tne Effect of some Inorganic Salts on Wumber of Soil Bacteria in the Soil Solution. Chemicals Days Beginning 16 36 60 II7 IT35 146 207 . Kel 1,393" 2397 1052 885 945 1345 395 460 NaNO, " 2475 2167 852 --- 645 400 105 NagHPO, " 1962 1940 1085 1380 1160 880 140 KCl + NaNO; " 2017 2525 1800 1480 985 972 506 KHpP0. " 1510 1425 19727 385 200 247 125 NaNOs + NagHPO, " 2057 2100 2100 1960 770 832 597 KH,P60, + NaNO, " 2635 2100 1985 2100 1042 330 497 Original Soil~801." 1415 2040 1160 465 612 485 272 *The number of bacteria in thousands per gram of oven dry sand. Table IX. The Moisture Variation in the Cultures Chemicals Days 18 36 60 117 #2+:132 «#146 ~=« 207 KGL 8.3% 8.3% 8.1% 7.1% 7.4% 7.4% 9.0% NaNO, 7.5 8.4 7.5 7.0 6.2 7.4 8.4 NagHP0, 7.9 8.1 7.3 7.2 %4 8.1 8.6 KC1 + NaNO, 8.0 8.1 7.9 7.2 7.7 8.0 8.6 KHgP0, ” 8.1 8.4 7.6 6.9 7.0 7.1 8.9 NaNO; + NagHPO, 7.8 8.5 7.3 7.3 6.8 7.6 8.4 KHgP0, + NaNO, 8.0 8.4 7.7 7.6 7.3 8.0 8.8 Average % Moisture 7.9 8.2 7.6.7.2 7.9 17.7 48.7 = 30 - most beneficial influence as follows: 4} KHgP0, + NaNO, 2,635, 000% 2) NaNO, 2,475,000 3 KCl ” 2,397 ,000 3) NaNO; + NagHPO, 2,057,000 KCl + NaNO, 2,017,000 NagHPO., 1,962,000 $) KHgP0., , 1,510,000 Original soil solution 1,415,vu00 *% Number of bacteria per gram of oven-dry sand after eighteen days of incubaibn. When the effects of various salts on duration of bacterial development are compared, they might be classed in four groups as to toxicity: | 33 KCl becomes toxic between 18 to 36 days. 2) NaNOs, NagHPO,, KCl + NaNOg, and original soil Bolution became toxic between 36 and 60 days. 3) KHgPOq inhibits growth from the start and be- comes toxic between 6Qartd 117 days. 4) NaNO, + NagHPO,, and KHgPO, + NaNOg become toxic between 117 and 14 days. To proceed with explanation of the nature of the Ca.ses imading to specific action of different salts on bvac- terial develppment, firstof all, it is important to study the secondary effect, nemely the changes of reaction in the culture medium as shown in Table X. | Analyzing the data on Table VIII and X and comparing the curves in Figure 3, there is strong indication that the various salts haveadifrerent effect upon the changes occuring in the sand cultures and that they thruough these changes, in- fluence the bacterial development. During the first eighteen days there was a marked increase nf number of bacteria in all ‘cultures except those with monopotassium phosphate and the checks. It has been shown above when discussing the effect of sulphuric acid upon soil bacteria, that the most suitable reaction for 99.4 percent of bacteria was N/1000 alkline (-1,0°Fsg — Table X The Changes of Reaction in Cultures to Which Varbous Inorganic Salts were Added. Days 18 36 60 117 132 146 20% Chemicals Very Very Sl. KCl Neut. Neut. S1l.Acid Sl.Acid Acid Acid Neut. NaliOg Alk. S51. £1 .Acid " Acid " " Alk. | NagHPO, " Alk. Alk. Alk. Alk. Alk. Alk. Sl. Sl. Sl, KCl + NaNO; . Alk. Neut. Acid Acid Acid Neut. Sl. Sl. KH2PO4 " " Alk. Alk. Lieut. Acid Acid Alk. NaN@, + NagHPO, " " " Alk. Alk. Alk. "* . Sl. Sl. 81. KHgPO,q + NaNO, " " " Alk ik Alk. " Sl. Original Soil™Sol. " Alk. Acid Acid Acid Acid Acid Check Neut. Neut. Neut. Neut. Neut.Neut .Neut. When the KHgPQ, chltures are considered it is not the alkalinity of KHgP0, that prevented the growth of bacteria, because KH2gPQg is acid in reaction, consequently the retailing eftect of KHgPQ,4 on bacteria is probably on account ef some ch nge eccurring in the culture before this salt became toxic. Shmxz The analysis on the eighteenth day shows that the culture had become alkalin in reaction. Whether it is a purely chemical change or whether the Change is due to bacter£al activity we are not prepared to answer definitely. The chagge in the reaction of cultures was unexpectd and no preparation was mede for its study, As far as results are concerned, the reation of the medium appears to be a great factor in the development of bacteria. ‘ihe changes in the reac- tion of the cultures are gradual, they reach a certain point and begin to gottheopposite direction as is the case with KCl, NaNo,, - 32 - KHgP0,4, and with the combinations of these salts. On the @ighteenth day some ciltures are neutral and some are alkalirm They gradually become slightly acid and after period of 207 dgys of incubation turn back to a neutral or an alkalin condition. H owever, in the case of NagHPO, and its combinations the reac- tion is always alkalin which probably is because this salt is alkalin in reaction. On a whole it is noticeable that the com- bination of two salts provides a better environment for bacterial development than a single salt and that the change in reaction of the cultures with these salts is not as rapid as with some of the single salts and consequently the toxicity of the culture is delayed. It, however, has not been explained why one salt become toxic earlier than another or why the combination of naNQOg + KH3P0, and NaNOg + NagHPO, support bacterial life several times linger than NaNO, or the other individual salts. It does not appear to be a question of the lack of food supply in the cul- tures because there was no waste of the mineral substances, unlessthere was a volitilization of ammonia, add the number of bacteria was not great enough to assimilate alii the nutrient constituents in the medium. The reaction of the cultures con- taining NaN@g deeame acid in less time than those containing two salts. This, in pazt, accounts for the greater number and the longer persistence @f bacteria $n the cultures to which several seits were eadded. - 33 « General Discussion The experimental work on this study of the effect of reection on soil bacteria in.the soil sblution brought out fac t® to abuw the deleterious nature of acids. Alkalin reaction of the soil solution becomes inhibitive to bacterial growth if OH-ion concentration was above N/L@00 (-1.0°F.S.). The number of bacteria which was not killed either in N/1200 hydrochloric or in N/1200 sulphuric acid was insignificant, namely, about 2 per- cent. The reaction with H-ion concentration of N/2860 sulphuric acid was inhibitive to 46.5 percent of bacteria when the cul- tureswith this reaction were compared with N/1000 alkalin reaction cultures after the eighteenth day of incubation. Comparing the miftfmrliterature on the"€oxic limits" of acids towards plants, one finds divergent reports, indicating the difficulties in determining what is so toxic limit for a certain plant. This is brought out clearly by the figures represented below by Loew (68), Heald (39), Cameron and Breazeale (40), Hertwell and Pember (41), asd Gedroitz (42): Loew Heald Cameron Hertwell Gedroitz Braezeale Pember Indian Corn Barley Seedlings Seedlings Hydrochloric Acid N/512 4N/3200 wN/3000 N/5000 N/1250 Sulphuric Acid N/512 =N/3200 N/3000 N/5000 W/1250 results With exception of Hartwell and Pember’s(41) the toxic limit of acids to corn and barley seedlings approach the point at which the greatest number of soil bacteria were killed. The lowest H-lon concentration (N/2860) inhibited growth of 46.5 percent of = 34 @ bacteria which ie close to Cameron and Breazeale's “toxic limit” for Indian corn seedlings. These data show distinctly that soil bacteria in general are more sensitive to acid conditions than some of the agricultural crops. Consequently the neutrali- zation of the acidity in soils would permit of the bacterial development with which is associated the production of soluble salts used for plant growth. When sand cultures containing alkalin soil solution are kept for a prolonged period, the reaction of the cultures Ehag changes from alkalin to acid. This phenomenon is commonly ob- served in test tube with some carbohydrate nutrient solubion, when fermenting organisms are present. In soil solution such explanation probably will not be sufficient to account for this change, because the soil solution came from soils one of which did not contahn large amount of organic material, while the other ceme from a rich soil. However, the changes in reaction in both were similar in nature. The changes in reaction from acid to alkalin could not be due to leaching out of bases as it occurs in fields, neither would it be due to adsorption of bases, beaause first, the cultures were kept in resistantegtass flasks, second, the quartz send has a very slight adsorbing power (14). The disassociation and the recombination of ions is probably the result of becterial activity. Undoubtedly chemicel changes might occur in soil with out bacterial intervention, yet the data at our hand hardly justify such admission. Two different soil solu- tions to which thymol was added to prevent bacterial development were kept in resistent glass flasks under rubber stoppers for four months. The solutions titrated the following degree of reaction: -35- At beginning After four months Solution A. Neutral Neutral Solution B. Alksalin (0.21% normal) Alk. (0.217 % nor.) To test whether added salts will produce change in reaction after standing in papafined flasks under rubber stoppers the above solutions were treatéd aes follows: Reaction at beginning Reaction after 54 days Solution A + KCl Neutral Neutral | " + NaNOg Slightly slkalin Slightly alkalin " + KCl . | " " NaNO, " «B+ KCl Alkalin(0.016% N) Alkalin (0.02% N.) " C Unal- Alkalin(0.044% Nz) - Alkalin (0.04% N.) tered " + NaNO, " (0.04% N.) " (O0F046%N. ) " + KHaPO,Acid (0.4% N.) Acid (0s2% N.) The results ontfewhole indicate that in absence of bacterial life end without influx of oxygen if there was any change it could not be detected by titration with N/100 sulphuric acid and N/100 alkali. The cagses which probably enter intthe alteration of the reaction of the sand cultures in presence or absence of added mineral salts might be several. In the process of bacterial growth one of the ions might be used by the organisms in excess of the other thus setting free the other ion which will influence the reaction of the medium. If potassium is assimilated in ex- cess of chlorine from KCl the reaction will become acid. When several salts are present in the culture the assimilation of some of the ions by the microorganisms can lead to a recombina- tion of the remaining ions and thus produce ea new condition in the medium which might be toxic to the microorganisms. To a certain limit the number of bacteria is associated with the change of the reaction.InIheearly part of the experi- ment the increese in the number of bacteria and their consequent decrease wes parallel with the change of the reaction of the medium According Fred end Hart (32) monobasic potessium phosphate caused enenormous increase in number of bacteria in the first two or three days and a sudden drop followed after the fifth day. This, in part, might account for the low number of bacteria in our cultures with KHgPO, efter eightéenth day of incubation. However, there is no pérceptible change in reactim of the culture medium that would suggest the cause Bor such Sudden drop. The effect of various inorganic salts added to the B80il solution does not bring out a strong indication of their Bpecific influence on soil bacteria, ymxt under conditions of our experiment. Yet, they undoubtedly show, first, that the addition of a sait delays the change in reaction of the soil solution; second, that a two or three salt combination supports & greater number of bacteria for a longer period than one salt. - 37 - Some Morphological and Physiological Characters of the Bacteria Isolated fron Cultures with n/1200 Acid Reaction Morphology 123 4 5 6 7 8 Form (Rod + + + + = + + + (Spherical - = = - * 5s + = Endospores + + + + = + + + Lotility + + ¢ |= + = + + Pigment - + + s= + #2 © «@ Relation to oxygen Aerobic - - - - - Bacultive + + + + + + + + Ammonification, Nitrification and Denitrification Studies of the Organisms. | : | No. Dunham's Solution Synthetic Media of : Z -_ NOg | NH, . NOg-| NH, | WNOg | NHy NOs | NHg| | : J . oo | 2 - - - - a — + | 5 - |) - - - + + - + | 4 - |. - - + + + + | . 5 coe le - - - + + | | 6 - - - - + + - + | 7 - - - - + + + + | g _ . ae _ _ . _ | i L L « = Negative. + = Positive. # = Doubtful. = 38 - Sumnary . The literature reveals an important relationship between bacterial growth and the reaction of the medium. Molds preferaslightly acid medium. Bacteria vary in their power to grow upon slightly acid media. Some, like Bact. lactis acidi, develop when the reaction of the medium is about .3 percent acid, while a majority of putrifactive bacteria preferaslightly alkalin reaction. The work with send cultures of soil solution ex- tracted by the oil pressure method, reveals that the reaction of alkalin soil solution gradually changes from alkalin to neutral and later to acid. With the change of the reaction of the sand cultures of the soil solution, the number of bacteria changes pari passu with an avorupt drop when the re- action becomes acid. When the reaction of the cultures was made N/12v00 acid either to hydrochloric or sulphuric acids, only about