THE. EFFECT OF OXYGEN DIFF‘JSION RAE ON NETRMCATION Thesis ‘or ”no Degree of M. 5. MECHIGAN STATE UNWERSITY Gerald H. Brandt 1960 “i Michigan State 31 Umversity LIBRARY THE EFFECT OF OXYGEN DIFFUSION RATE ON NITRIFICATION BY Ge rald H. Brandt A THESIS Submitted to the College of Agriculture of Michigan State 'University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Soil Science 1960 ACKNOWLEDGMENT The author wishes to express his sincere appreciation to Drs. A. R. Wolcott and A. E. Erickson for their untiring assistance in conducting this study and preparing this manu- script. He is also extremely grateful to his wife, Evelyn, for her continuous support and endless assistance. :4: >3 :1: 3'; >5: >§< :',< 3'5 :3: z}: 3": 3'5 :1: :1: :1: ii THE EFFECT OF OXYGEN DIFFUSION RATE ON NITRIFICATION BY Gerald H. Brandt AN ABST RACT Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Soil Science 1960 ppppppp QXWM ABSTRACT Oxygen diffusion rates in soil, measured with a platinum micro- 2 min"1 to 57 x 10"8 gms electrode, were varied from 1 x 10"8 gms cm' cm’z min"1 by varying aggregate size and moisture tension. Nitrate and ammonium nitrogen were measured frequently over a sixty-day incubation period. Late in the incubation period, accumulated nitrate decreased sharply above and below an optimum oxygen diffusion rate of 30 x 10'8 gms cm’zmin'l. No nitrate was found below a threshold of 3 x 10"8 grns cm”z After an initial delay, ammonium disappeared at uniform rates, min". except for short periods of net ammonification in some treatments. Ammonium disappearance curves were used to estimate delay periods and maximum rates of nitrification. Both maximum nitrification rates and delay periods increased with increasing oxygen diffusion rates, except in totally saturated soils where nitrification was indefinitely delayed. - In small aggregate separates used to achieve low oxygen diffusion rates, reductive or assimilative losses of nitrate occurred. - In-larger separates used to achieve high oxygen diffusion rates, low moisture tensions were employed, giving rise to saturated micropores and locally anaerobic conditions within the aggregates. Reduced avail- ability of oxygen to these microhabitats was not detected by the micro- electrode. Nitrate accumulation in these treatments was erratic. iv TABLE OF CONTENTS Page INTRODUCTION ......................... 1 LITERATURE REVIEW ..................... 2 MATERIALS AND METHODS .................. 7 General Requirements ................... 7 Soil ............................. 7 Addition of Ammonium .................. 9 Moisture Adjustment and Control ............. 9 Incubation ......................... 12 Measurement of Oxygen Diffusion ............. 12 Chemical Analyses ..................... 14 RESULTS AND DISCUSSION ................... 16 Oxygen Diffusion Rates ................... l6 Nitrate Nitrogen .................... 16 Ammonium Nitrogen ................. 22 Soil Reaction ..................... 22 Nitrite Nitrogen ..................... 27 Total Organic Nitrogen ...... . ........... 27 Total Inorganic Nitrogen ............ . . . . . 28 Average Oxygen Diffusion Rates ........... 32 High Diffusion Rates ................. 33 Synchronous Fluctuations of Ammonium and Nitrate . . . 37 Unamended Samples .............. . 3? Amended Samples ................ 40 Estimation of Nitrification Rates and Lag Times. . . 47 Lag Time and Oxygen Diffusion Rates . . . 50 Maximum Rate of Nitrification and Oxygen Diffusion Rate 52 Threshold and Critical Levels of Oxygen Diffusion. . . 54 Practical Implications ................ 58 Criticism and Evaluation of Methods . . . . ..... 60 SUMMARY AND CONCLUSIONS . . ......... . ..... 64 BIBLIOGRAPHY . ” ................ 67 APPENDIX . . . . . . ............ 71 LIST OF TABLES TABLE 1 10. 11. 12. 13. 14. 15. 16. . Nitrate nitrogen in amended soils. . Nitrate nitrogen in unamended soils. . The pH of unamended soils . Description of treatments used to establish varying levels of oxygen diffusion for support of nitrification in soil. OOOOOOOOOOOOOOOOOO . Oxygen diffusion rates in amended soils ........ . Oxygen diffusion rates in unamended soils . AInmonium nitrogen in amended soils. . . Ammonium nitrogen in unamended soils . The pH of amended soils. . . . ........... Organic nitrogen in amended soils after 58 days in incubation. . . . OOOOOOOOOOOOOOOO Organic nitrogen in unamended soils after 58 days incubation. . . . ...... Tabulation of pH values obtained from 1:1 soil to water suspensions from each tube incubated. . . . . . Tabulation of measured oxygen diffusion rates ob- tained from each incubated sample with time. Tabulation of nitrate nitrogen values for each incu- bated sample. . . . Tabulation of ammonium nitrogen values for each incubated sample with tiIne. Moisture tension data . vi Page 17 18 19 20 23 24 25 26 29 29 72 74 76 '78 80 LIST OF FIGURES FIGURE 10. ll. 12. 13. Capillary tube positioned in a soil column for release of trapped air during water addition. . . . . . . . . Modified platinum electrode for measuring oxygen diffusion rates of soilincubated in test tubes ..... Changes in total inorganic nitrogen with time . . Changes in total inorganic nitrogen with time . . .- Ammonium and nitrate nitrogen appearing in the unamended samples with time ....... . . . . . . Ammonium and nitrate nitrogen that occurred in the amended samples, treatments 1 and 3, with time . . Nitrification patterns in the amended soils, treat- ments 5 and 7 ..... p .............. . . . Nitrification patterns in the amended soils, treat- .ments 9 and 11, with time ....... . . . ..... Nitrification patterns in the amended soils, treat- ments 13 and 15, with time .............. Lag times (t') as they were affected by oxygen diffusion rates ..................... Maximum nitrification rates as they were affected by oxygen diffusion rates ............... The relation of nitrate nitrogen accumulation to oxygen after 40 to 43 days of incubation . . . . . . . Relation of nitrate nitrogen accumulation to oxygen diffusion rate at 49 to 50 days of incubation . . . . . vii Page 11 11 3O 31 38 41 42 43 44 51 53 55 57 ' INTRODUCTION Nitrification, the microbial transformation of ammonium nitrogen to nitrate nitrogen is one of the important soil processes that requires oxygen. Attempts to quantitatively evaluate the nitrifying capacities of soils have indicated the need for measuring environmental factors, such as aeration. The relationship between oxygen dependent soil processes and soil oxygen have been studied heretofore in terms of partial pressure of oxygen in the ambient or soil atmosphere. The partial pressure of oxygen in a soil system gives no direct measure of the rate at which oxygen is supplied to reaction sites or micro-habitats in the soil. The platinum electrode, which measures oxygen diffusion rates, can provide data which was not previously obtainable in studies involving the relationships between oxygen supplies and certain chemical or biological processes. Oxygen diffusion rates in soils measured with the platinum micro- electrode have shown good relationships to crop yields in recent studies. Growth of plants in these studies was reduced by very short periods of oxygen stress at critical stages of growth. This re5ponse may be a direct result of inhibited root growth or it may be an indirect response to the inhibition of soil processes which require oxygen, such as nitrification. Diffusion rates are affected by the physical state of soils. Moisture content alters diffusion rates appreciably by changing the amount of water held in capillary pores and the thickness of water films over soil particles. - Structure and texture can greatly affect diffusion rates in a soil body by changing the number of large pores available for gaseous diffusion. The objective of this study was to quantitatively assess the relationship between oxygen diffusion rates as they are affected by these soil factors and nitrification occurring in soils. LITERATURE REVIEW Nitrification is known to be affected by many environmental factors. Among these factors is aeration or oxygen supply. The classic forerunner of all studies in this field was by Schloesing and Muntz (36) in 1877. After demonstrating the microbial nature of ammonium oxidation, these workers went on to show that almost as much nitrification occurred in soils at an oxygen concentration of 11 percent as at 21 percent. This study followed their observation that a decrease in nitrification occurred above a certain wetness, presumably because the closing of pore spaces by water reduced aeration. Work done by Gainey and Metzler (16) explored the relationship between oxygen concentrations and nitrification. Their results substantiated those of Schloe sing and Muntz and went on to show that soil air does not vary greatly in composition with depth in the profile. They concluded that conditions are rarely met where there is not sufficient oxygen potentially available to insure maximum nitrification. More recent studies by Amer and Bartholomew (2) confirmed the observation that decreasing the oxygen concentration from 20 to 11 percent has a negligible effect on nitrification rate. They also stated that a minimum level of oxygen concentration exists somewhere between 0. 2 and O. 4 percent where nitrification does not take place. The partial pressure of oxygen in the soil air is rarely more than 1 or 2 percent lower than that in the atmosphere, except in water logged soil or in heavy, compact subsoils. These data suggest that nitrification is seldom limited by the concentration of oxygen in soil air under normal field conditions. However, little is known about the extent to which oxygen supply to microhabitats in the soil is influenced by factors such as moisture content and aggregate size. Measuring oxygen content of soils in a meaningful and efficient way has been the goal of many soil aeration studies. Webley (48) presented a technique using Warburg vessels for the study of oxygen availability to microorganisms in the soil and discussed its possible use as an index of soil aeration. He suggested that the method is limited because it must be conducted under strictly defined laboratory conditions which are highly artificial when compared to those of the field. Russell (33) states that the importance of developing means of measuring soil aeration in terms of parameters that are meaningful needs to be emphasized. Diffusion is the most important physical process involved in the interchange of gases between the atmosphere and the soil. This fact has been recognized by many workers: Penman (28), Buckingham (6), Keene (18), Cannon and Free (7), Taylor (41), Raney (31), and Van Bavel (44). Accordingly, several workers have attempted to assess oxygen diffusion in soils, employing a variety of methods. A recent and promising procedure for measuring oxygen diffusion in soil by employing a platinum electrode has been described by Lemon and Erickson (24) and has been more completely deve10ped by VanDoren and Erickson (46). Oxygen diffuses to a platinum surface which is negatively charged with respect to an accompanying KCl bridge. Four electrons along with two or four protons are accepted by each molecule of oxygen according to the concept proposed by Kolthoff and Lingane (20). The electrons are donated by the platinum electrode which in turn draws current through a microammeter. The current is proportional to the number of electrons accepted by the oxygen. The diffusion rate can then be established by defining the area over which it occurs by applying Fick‘s diffusion theory as reported by Laitinen and Kolthoff (22). The following formula applies when diffusion of oxygen to the electrode is maximum, which infers that concentration of reducible material at the electrode surface is zero: . 3C 1tanAfxzo’tanAD(‘-é-;)X:O’t - current in amperes at time (t) in seconds H. H I n = number of electrons used per molecule of oxygen electrolyzed. The value 4, assumed by Davies and Brink (11) is used. ‘F = the Faraday, 96, 500 coulombs A 2 area of electrode in square centimeters fxzo, t = flux at the electrode surface at time t, or the number of moles of oxygen diffusing to the electrode per second at time t. D = diffusion coefficient of oxygen in square centimeters per second C = concentration of oxygen in moles per cc at a distance x centi- meters from the electrode surface t seconds after beginning diffusion (by closing the circuit). The equation expresses diffusion in moles of oxygen diffusing to the electrode at time t. This can readily be converted to g. of oxygen cm. ‘2 min. "l The influence of enviromnental factors on nitrification has been evaluated by numerous methods. Most involve quantitative comparisons between the factor being studied and static measurements of the amount of nitrate produced‘or the amount of ammonium lost after an arbitrary incubation period. More recently investigators have recognized the need for considering the dynamic nature of the nitrification process and have attempted its quantitative characterization in terms of the classical bacterial growth curve. The bacterial growth curve as related to carbon dioxide evolution has been discussed by Corbet (10). Similar attempts to express observed nitrate production curves mathematically have been made by Miyake (27) and evaluated by Pulley and Greaves(29). Quastel and Scholefeld (30) derived and discussed expressions for the oxidation of ammonium and nitrite using maximum rate values (K values) and discussed the significance of lag time. The use of the lag period and the maximum rate to characterize nitrification in soils has been discussed by Stojanovic and Alexander (40). A mathematical model for the quantitative prediction of nitrification rates in soils was proposed by Sabey, (it a_1. (34). In a given soil at a given temperature nitrification approached a linear function of the form: N=K(t-t') where: N = nitrate produced K = a constant characteristic for a given soil at a given temperature. t = timesof incubation t' = lag time. In a later study, Sabey e} a}. (35) found that both the maximum rate constant (K) and lag time (t') were influenced by temperature, whereas only lag time was affected by the number of nitrifying organisms initially present. The influence of temperature and numbers of nitrifiers on lag time and maximum rate varied greatly among different soils, indicat- ing that other factors not measured had influenced nitrification. The general usefulness of the basic mathematical model would depend upon quantitative evaluation of the effect of other environmental factors on lag time and maximum rate. Among factors to be considered would be temperature, moisture, pH, texture and aeration, or oxygen supply. These workers emphasized a point frequently ignored, and that is that ammonium should be supplied initially in quantities that will not become limiting during the period over which nitrification rates are measured. A difficulty frequently encountered in nitrification studies is the fact that nitrate formed from ammonium oxidation is not quantitatively recovered because of gaseous losses due to denitrification. Broadbent and Stojanovic (4) have studied the relationships between denitrification and partial pressures of oxygen. They concluded that denitrification rate was affected more by the quantities of nitrate and oxidizable carbon in the soil than by partial pressures of oxygen in the soils they inve sti- gated. Their findings are in agreement with those of Korsakova (21) who studied the reduction of nitrates by Achromobacter siccum and Pseudomonas aeruginosum and Meiklejohn (26) who studied aerobic denitrification. MATERIALS AND METHODS General Requirements Nitrification was quantitatively characterized in terms of lag periods and maximum rates observed during a nine-week incubation. Oxygen diffusion rates were effectively controlled by employing a range of aggregate sizes and various moisture levels. A preliminary incubation was used to establish effective diffusion levels and to determine the approximate duration of the eXperiment. Effective diffusion rates had to be maintained in a small volume of soil so that a large number of samples could be incubated and efficiently manipulated during the extraction of ammonium and nitrate. The system also had to yield reproducible oxygen diffusion readings within each diffusion range treat- ment for the duration of the incubation. Diffusion rates were adjusted in preliminary studies, to vary uniformly from 1 x 10“8 to 65 x 10“8 .2 min'1 by varying aggregate size and moisture tension. gms cm Reproducible means of two or three readings per sample could be obtained at a depth of 1 1/2 to 3 1/2 inches in 4 inch columns of soil contained in 20 x 215 mm test tubes. Soil A well aggregated Brookston clay loam, pH 5. 9, was used to obtain the range of aggregate separates shown in Table 1. It had been stored air-dry for a period of about four years. It was thought that a greater range in lag period might result from a soil stored for a long period of time, since initial nitrifying pOpulations would be low. ‘ Aggregate separates were obtained from this soil by sieving. Part of the larger aggregates were crushed in order to obtain adequate amounts of each size of separate. The soil used and the aggregate sizes Table 1: Description of treatments used to establish varying levels of oxygen diffusion for support of nitrification in soil. Treatment Aggregate NHi Moistuie Level Number Size (mm) Added cm=’-=* percent 1 4.00 - 6.00 Yes 10 42 2 4.00 - 6.00 No 10 42 3 2.00 - 4.00 Yes 10 47 4 2.00 - 4.00 No 10 47 5 1.00 - 2.00 Yes 10 50 6 1.00 - 2.00 No 10 50 7 0.42 - 1.00 Yes 10 57 8 0.42 - 1.00 No 10 57 9 0.17 — 0.42 Yes 35 42 10 0.17 - 0.42 No 35 42 11 < 0.17 Yes 400 42 12 < 0.17 No 400 42 13 0.17 - 0.42 Yes Sat. 65 14 0.17 - 0.42 No Sat. 65 15 < 0.17 Yes Sat. 60 16 < 0.17 No Sat. 60 >1: (NI-i4); SO4 added at rate of 400 ppm N. Uneven distribution in larger aggregates and probably chemical fixation produced an effective range of amendment of 220 to 320 ppm N. 31¢ a): Tension in cm of water. Table 16 in the appendix shows moisture tension and percent moisture for each aggregate size. employed corresponded closely to those used in a greenhouse experiment conducted by Lemon (23) to study methods of measuring oxygen content in soils. The range was extended to separates of smaller size for the present study so that lower diffusion rates at shallow depths could be obtained. Addition of Ammonium Ammonium nitrogen at the rate of 400 ppm was added as ammonium sulfate to one series of separates. A preliminary incubation extablished this as a non-limiting ammonium source for the conditions of the incu- bation. Mixing of the ammonium source and the soil was accomplished by first weighing the air—dry aggregates, adding a small amount of water and mixing to obtain uniform distribution of the water. A known amount of ammonium sulfate was added by spreading the soil out on paper and Sprinkling the ammonium source uniformly over the sample. The aggre~ gates were again rolled together, mixed and weighed to determine the amount of water added. Analysis of the treated separates before incubation showed erratic distribution of ammonium in the larger aggregates. The effective rate of addition of ammonium nitrogen in these larger aggre— gates was 220 to 320 ppm. The equivalent of 40 gm of air-dry soil was placed in 20 x 215 mm test tubes for incubation. This gave a column of soil 4 inches deep in each tube after gentle tapping. Additional water was added to achieve a predetermined moisture tension as described below. Thirty tubes were prepared for each treatment. Moisture Adjustment and Control Moisture tension curves were prepared for each separate size using tension plates and pressure membranes. At low tensions, determinations 10 were made at 5 cm increments to give a valid curve in that range, as most of the treatments were incubated at a tension of10 cm of water. By use of the moisture tension curves, additional water was added to the soil in the test tubes in amounts calculated to establish specific moisture tensions for specific separates, as shown in Table 1. Two treatments among the small separates were moistened to 32 percent moisture content, which gave a tension of 35 cm in the 0.42 - 0. 17 mm and 400 cm in the < 0.17 mm aggregate size. These‘corresponded in moisture content to the 10 cm tension treatment at 32 percent moisture in the 4. 00 - 6. 00 mm separate. A difficulty encountered in preliminary studies was the entrapment of air when water was added to the soil in the test tubes. Air bubbles formed which disturbed the uniformity of the soil column and also inter- fered with the rapid movement of water into the lower portions of the soil column in the smaller aggregate separates. It was found that a capillary tube could be inserted down the side of the tube to bleed air from the bottom of the soil column (Figure 1). A teaspoonful of vermiculite on the soil surface prevented slaking of surface layers as measured quantities of water were added slowly from a burette. This method of adding water proved to be very satisfactory. ‘ After wetting in this manner the capillary tubes were removed, the holes filled with short pieces of glass rod ‘ and the test tubes laid horizontally for a time to allow for moisture equilibration. They were then placed in the incubator in a vertical position. Moisture loss from the tubes was prevented by capping them with polyethylene. The oxygen concentration in the atmosphere above the soil in polyethylene-capped tubes was measured in selected tubes with a Beckrnan oxygen electrode. This electrode gives instantaneous readings from atmosPheric oxygen which can be converted into percent concen- tration with preper calibration. 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In amended soils and generally in unamended soils, the highest nitrate levels were found in the next-to.- smallest aggregate size adjusted to 35 cm water tension (Treatments 9 and 10). The nitrate values for these two treatments were signifi— cantly higher than for treatments involving larger aggregates and significantly higher oxygen diffusion rates. The same inverse relation— ship between oxygen diffusion rate and nitrate accumulation was found in amended soil (Tables 2 and 4) when the smallest aggregate size adjusted to 400 cm water tension (Treatment 11) was compared with the three largest aggregates adjusted to 10 cm (Treatments 1, 3 and 5). These anomalies indicate that other factors in addition to oxygen diffusion rate were influencing nitrate accumulation in these soils. Because diffusion rate was controlled by moisture level and aggregate size, we might suspect that these anomalies may be the result of associated variations in the controlled physical environment, such as the thickness of water films, the degree of saturation of micropores in and around aggregates, as well as with the ratio of surface area to internal volume of aggregates of varying size. Further consideration will be given to these possible factors at a later point. Nitrate nitrogen was higher at the end of the incubation than it was at the beginning of the incubation, except in the saturated treatments and in treatments 8 and 12 which were unamended samples adjusted to 10 and 400 cm tension, respectively. However, the observed increases in successive samplings could not be clearly interpreted in terms of a normal nitrification curve (35). There was evidence of alternating appearance and disappearance of nitrate. This cyclic tendency was not synchronous between treatments or between amended and unamended series of the same treatments. Because the cyclic fluctuations in the amended samples were not simultaneous with those of the corresponding unamended soils, nitrate values calculated on the usual basis of "percent added ammonium nitrified" (39) were without meaning. Ammonium Nitrogen Ammonium levels generally decreased in the saturated amended soils (Table 6) during the course of the incubation without any correspond— ing increases in nitrate (Table 4). In other treatments, changes in ammonium level generally complemented the trends described for nitrate. 1n treatments and on sampling dates where nitrate was low, ammonium was high, and vice versa. This was true also for unamended soils (Tables 5 and 7?. The relationship was not linear, however. Treatments with high oxygen diffusion rates (Table l), tended to be significantly higher in ammonium (Table 6) than treatments with lower oxygen diffusion rates, except in saturated soils where high ammonium levels were associated with low diffusion rates. Fluctuations in ammonium up to 150 ppm in a period of less than a week were observed. Nitrate showed less extreme fluctuations, varying at the most about 50 ppm from one sampling to the next. As was true in the case of nitrate, there was no synchrony in the cycles of ammonium appearance and disappearance among treatments, nor between amended and unamended series of the same treatment. Soil Reaction Determinations of pH made at the time of sampling gave a rough estimation of the rate of nitrification occurring with each treatment. Sampling frequencies were determined occasionally using pH trends since it was not always possible to analyze the extracts at one sampling date for ammonium and nitrate before the next sampling day arrived. Tables 8 and 9 are presented to show the trends which occurred in soil reaction with treatment and with time. 23 .Uoflom mcflmamw zoom GEfiB cam on “sonommwp twang-mognmwm nos who? “manomQSm Hmofion-mgmH-m mad-mm may 5;? modfim> no 6 .o .o. rm 3.3m .23 2.3 ms: 93 ~.mm 2.3 93» mg: m3~ - aonmfimeegmeqfim Mai: .33 - amen-N - Bag-m. nan-3 nomm 33 253 m3 2.v 4mm 3 $3 $3 - nmmwm - 38:3 032mm name-a. 33 30mm 2m 3:2. .st .2 082 33 33 no.2 3.2 32 032 £33 253 433 Sm 2.v 03 2 332 Q32 302 8.32 no.2 3332 833 053 8.2m 8&3 3m 3:2. mm o 0; £2 no.2 nan-NM $3 332 032 £33 «33 8&3 2m 92-3. 2 - 332 .52 or: 32: m2~ €me 85mm 38m 8.03 m2m 3N N; S m 252 Q22 .53 £3 «mom J2 «Sm nm3~ oamm .ommm 33 TN 2 m 03 062 as: nmmmm mmow nmmom amom «mi owom 33mm o3 33 S 2 . he” z-Ez 2.3 3-3 mm-SEWmNW 8-3 3-3 3-3 3-3 3.3 3-3 2.: o Afifivmofim Eu 5.“ oudmoumwm G335» Hung: cog-monsoon mo 9an mo swam-n Hon-m3 «Goa GofiQCUmoQ undo-AH. .mZOm popcoad a“ flowchfid 8.9302224 .0 3an 24 32392-3 flodo mi??? 63”qu 0.0m um. noouow-fip afic-moflwdwfim HOG whoa, “manomfldm Hooflofl-mflmH-m mos-3m 02$ Hafiz, nova-2w “m .36 .o .3 .-m |.U on: 3.2 0.3 ms. o.m as 20 ms me as message 3356 a3 3... .3. m3 - m3 m3 m3 m3 mom 2 2 .v saw 2 m3 m3 .- do... - m3 8.3 nEN... 32m ona3 3 3:2. 5% 2 hm .5 m2 as as us so2 33m 33 33m 2 2 .v 03 2 now now 0:. am «.3 up 03 33 8:... 333 3 3:2. mm 2 of non am he do 0.3 86m 3m 33 95$ 3 o .2-3. 2 w pm or... no 3 m2 3.2 32 2U2 3% 3m 2 N; 2 .w .23 n... no 05 m2 52 332 €an Em... 33... 2 3-... S 3 3 hm 3 n... m3 03 32 3mm 3.“. 3m... f 33 S N z-fiz 2mm 33% mmém 33mm 3.3 3-3 3-3 3-3 3:3 mm .3 2.32 o -AEEV «.36 Eu 5 H. - . ouomonwmm cowmcou Hongc GOG-onobmfl mo mtg-mm mo owns-Q Hon-m3 . once Coflmwnomofl sumo-HH- .mfio.m popCoEmcs S.“ ammonia Edd-208$ .b van-QB 25 0 0 2.2 -.. mg -- 0.0 o p 2.» 0.0 20 2.v Jam 02 0.0 0.0 -- 0 0 -- o.» o p o h 0.0 0.0 ~0.-2. 2mm 02 -- MW.0 0.0 0.0 0.0 0.0 N.0 2.0 0.0 0.0 p2.v 000 22 -- ~10 N0 0.0 210 0.0 0.0 2. 0 0.0 00 3:2. 00 0 0.0 0.0 0.0 0.0 .3. 2.0 2.0 0.0 0.0 0.0 0.2-3. 2: p 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 m0 m0 0-2 02 0 ~10 0.0 0.0 0.0 0.0 0.0 20 0.0 2.0 ~10 TN 02 0 N0 0.0 0.0 0.0 0.0 0.0 20 0.0 2.0 2.0 0-2.. 2 2 00-00 00-00 2.0-00 00-00 00-02. 00-02. 00-02. 00-2.0 3.00 02.22 2860 0320 .80 c2 . Ll mudwmnmmm 220.3023 Hun—8.522 220363-9022 mo 0.0-mm mo mmamu 23.95 “dug 22330.3me $2.“.qu .mfiOm Umfifimgd mo mm 003. .w mzmp 26 o;- -:..-. 0.0 0.0 o.h 02.0 w.o SJ Jam 2: 0.» ..-.. 0.0 0.0 0.0 0.0 0.0 00-2. Jam 2.2 «2.0 22.0 N.© 20:0 béw v.0 w.© NIH.V 00¢ NH 0.0 0.0 20 0.0 00 N0 N0 00-2. 00 02 2.0 42.0 m0 m6 mb 0.20 0.0 0.2-2.. 02 m 0.0 N6 2.20 0.0 0.0 2.0.0 N0 NJ 02 o 2 0.0 m0 w.m 2.20 m0 m6 v.0 .v-N 02 2V n.m >6 w.m m.m 2.“ 2.0 m0 0:2. 02 M 002.00 021.02. 2.2102. 02.32. 00.20 02-00 02-22 2882 0320 Eu :2 . mudwmhwmm 2.200me0 Hmfiafin 2203206250222 20 0.3mm mo mmcdh .2395 22de . :oflmfiuomofl $2.828 320m Umwamamas mo mm @228 .o van-m8 27 The pH of the amended, unsaturated samples generally decreased from 6. 3 to 5. 2 during the incubation. The saturated amended and unamended samples increased in pH to about 7. 0 during the incubation because of the anaerobic environment produced by this treatment. - In unamended soils, this appears to have been due to an accumulation of ammonium (Table 7). In the saturated soils to which ammonium sulfate was added, there was no net accumulation of ammonium, although ammonium levels remained high throughout the experiment (Table 6). Here the increase in pH was due to reduction of the associated sulfate to sulfide which appeared as a black precipitate distributed in a mottled pattern throughout the soil. ~ A strong odor of H28 was detected when these soils were susPended in the acid extractant. The blackening of lead acetate papers confirmed the presence of H38 in the gases liberated in the extraction flask. The unamended, unsaturated soils showed small pH variations with increasing incubation time and had values at the end of the experiment that were close to the pH of 5. 9 observed at the beginning. Nitrite Nitrogen A qualitative test for nitrite was made on some samples during the beginning of the maximum rate phase. Occasional samples among the saturated soils showed traces of nitrite; however, the quantities found were less than two parts per million. Little nitrite would have been expected (39) at these pH's. Total Organic Nitrogen Organic nitrogen determinations were made on some samples during the latter part of the incubation, period. Amended samples were signifi- cantljr higher in total organic nitrogen than were unamended samples, 28 as may be seen in Tables 10 and 11. There is insufficient evidence to show whether this was due to an increase on the part of the amended samples or a decrease in the unamended samples. An analysis for organic nitrogen was not made on the soil before incubation. Total Inorganic Nitrogen Changes in total inorganic nitrogen (ammonium plus nitrate) are represented graphically in- Figures 3 and 4. All treatments in both amended and unamended series showed evidence of inorganic nitrogen loss during the later stages of incubation which are shown as ”mineral N lost" in the histograms. Nearly all treatments showed cyclic patterns of loss and recovery of inorganic nitrogen at different stages of incubation. Amended soils with oxygen diffusion rates below 20 x 10"8 gms cm“2 min'1 (Treatments 11, 13, 15) suffered consistent losses of inorganic nitrogen and ammonium from the beginning of the experiment. Both the amended and unamended soils in this diffusion range experienced an initial loss of nitrate. In the unamended soils (Treatments 12, 14, 16), this early loss of nitrate may have been due to a direct reduction of nitrate to ammonium, since the increase in ammonium during the first 18 days was approximately equivalent to the decrease in nitrate. In the amended soils, the loss of nitrate during this period contributed to a net decrease in total inorganic nitrogen. The saturated soils of this low diffusion group (Treatments 13 to 16) had oxygen diffusion rates which were generally below 10 x 10"8 gms cm"2 min-1. In these soils, only transient small amounts of nitrate were detected in samplings after 18 days, although additional losses of in- organic nitrogen occurred. The fact that detectable quantities of nitrate were found at various times in these treatments suggests that mineral nitrogen lost may have passed through the nitrate stage. In treatment 11, Table 10. Organic nitrogen in amended soils after 58 days incubation. 29 Alnount Over Hydrolyzable Non-hydrolyzable Total Treat- Nitrogen Nitrogen Organic Unamended ment PPM PPM Nitrogen Soil PPM PPM 3 1335 535 1870 282* 5 1215 630 1845 180* 9 1240 555 1795 140* 11 1255 570 1825 195* 4: - Significant at 1 percent. Table 11. Organic nitrogen in unamended soils after 58 days incubation. . Hydrolyzable Non-hydrolyzable Total Treatment - Nitrogen Nitrogen Organic PPM PPM Nitrogen PPM 4 1120 468 1588 1115 550 1665 10 1145 510 1655 12 ,1145 485 , 1630 30 600 Treatment 1 and 2 5- Treatment 3 and 4 I NH4-N 4-6 mm aggreg. 2-4 mm aggreg. I NO3—N 500. 10 cm HOH 10 cm HOH I. 551511511“? ODR = 40 ODR = 38 05 400- V C‘- .‘ (D n on g 300. , j ‘. . E 5“ '1 ‘3 53 f 5 ‘ "f 1.; 75 5 L1 2 200 .. :. .. -‘.- ; 04 "a 3. 3; 5 .2.- o- . '1.- ;é‘ .i~f e . 5? i 3?. f. 5.? ., if if: i‘ *- 5 7 .' .d‘ 0_ .Wm .. 0 14 28 35 4O 43 4- Days of Incubation Days of Incubation Figure 3—a Figure 3-b Treatment 5 and 6 Treatment 7 and 8 1-2 mm aggreg. .42-1. 0 mm aggreg. 500 . 10 cm HOH 10 cm HOH ODR = 33 ODR = 25 400 - . G .77 Q) ,. :4“. . 30° 3: - .- -» - i3 {1, +4 . a: ’5‘ n - . ‘5' i 14 ‘ '2 ‘ a 72 71 M r; ‘. 13‘ «'4 “ if: 3. L. ’3 [- H " 5:. y? k; ' ; . .. ' E 300 >4 " f; " 5' I :1 E‘- . Fa ' 35. '5‘: 53 5 . '2! ' .5‘ ‘11 i." a- . ES ,: 5 .- ii i ,_ i , Z, i‘I‘. :- * 1';- fi . ' r . 100 . :{2 ‘t; E“ .5 1 . -. ' E’ -. ..: 5‘ 55 ‘- 3“. 1'o I *4 i 5'." I" 1 ‘5 59 ~ 01428354043464954576201283540434854576368 Days of Incubation Figure 3-c Figure 3. Changes in total inorganic nitrogen with time. 4; Days of Incubation Figure 3-d The short bars represent unamended soils (even numbered treatments). Oxygen diffusion rates (ODR) are averages for both amended and unamended soils over the entire incubation period. PPM Nitrogen PPM Nitrogen Treatment 9 and 10 .17-.42 mm aggreg. 35 cm HOH ODR = 27 :' . ' f. 5.5 - .300. ,' ,. ,' v; .5 15 C" 5.; g 5. 5; .1 a .556 53‘: ‘ r25 5‘ 5 5' 55 5 5 5:15 “ 5 "4 :1 5 " '5 5 200 555 ' 5 5" ' 5 557: ' " 1:. . 5'". 5 ". . " ' .‘ 94 :‘5 5' :- ‘ I5 5:25 5 , I ‘ . ' . 51!! 018 25 34 4144 47 50 53 Days of Incubation 5. 5 is ‘I . Figure 4-a Treatment 13 and 14 .17-.42 mm aggreg. saturated ODR: 6 500'- 400 . \ '3! 31‘ - _ , 5 -‘ 4 =1 ~ 0 ‘ A. ~ , 2‘; u.' f "5 4.. 5" "5 I. '31 200 .. .1 a» a e. . - 3:5 5", "5 ‘5‘ "f 5.5" ' '- . .i: ‘ . .3. . : ‘ i " = .‘ 1 .' .5 'J ‘1' 1‘ 5'. - ' J' "'1'!" ‘5‘ a) ta: c} .; ‘§ 1. ‘1‘ ' 5 1, 'n c. l“. 5.,5 5 5"“ 5“: L55" I“? f” .1 5“ '5'; 10 0 3‘ If '5? u; ' E 3;; ' . _ . ,“ “'1‘.” . c "E! l. . T- .fi" . I _V ‘5’ _ 36 43 5O 57 64 90 O 18' Days of Incubation Figure 4-c Figure 4. Changes in total inorganic nitrogen with time. 31 Treatment 11 and 12. <. 17 mm aggreg. . 400 cm HOH ODR : 12 ' "4 :2!’ 3:3“ a:— E . 1‘ t.“ ‘4 I .4..- £1 11 '_ 5‘ ':_.n15 1 ‘ "' --- ‘wut: ’ a... r ._ TV'.’ 'f‘\~“;t_::: ‘ ';' "‘ 233.59:th - 3 ~13"! I.» . 1; r‘. u a .1.“- , 1 -W" '4‘” - x v.4 5" -0 4 . .‘ -. .-,;- wrap? . . ‘. _. 1_ ‘ ’ ‘. ‘ . It. WV. ’ - 1' ”a -_ .1 o" _ . _ . 1- . .. ' 25 34 4144 47 50 ‘33 58 Days of Incubation Figure 4-b Treatment 15 and 16 <. 17 mm aggreg. saturated ODR = 5 . w ~’- l. I 0;" 5 fig --‘:; a": 1’le ' k: 53.15 J. l“ E ‘9. 55' . 1 5- a‘ r 5 "5" ' ... ‘v .. ‘ )IT' ~95 .4311." 2:5: ' 1:55.: .- ‘ \_- . P «('34 ’4 555 '5' . § 5 25 36 43 50 57 64 90 0 18 Days of Incubation Figure 4-d The short bars represent unamended soils (even numbered treatments). Oxygen diffusion rates (ODR) are averages for both amended and unamended soils over the entire incubation period. 32 with oxygen diffusion rates generally between 10 and 20, conversion of ammonium to nitrate proceeded smoothly and inorganic nitrogen was apparently lost at the expense of nitrate. This strongly suggests that inorganic nitrogen deficits observed in these soils were due in part, at least, to denitrification. It is conceivable, however, that a portion of the nitrate may have been assimilated by microorganisms and retained in the soil in organic form. Average Oxygen Diffusion Rates Oxygen diffusion rates were somewhat higher (20 to 30 x 10'8 gm cm‘z min'l) in Treatments 7 and 9, and initial nitrogen deficits were not as pronounced. In fact, there was evidence of early net release of inorganic nitrogen. Losses of total inorganic nitrogen observed at later stages of incubation in treatment 7 were much greater than the losses observed in treatment 9, which had approximately the same measured oxygen diffusion rate but a lower moisture content (higher moisture tension). The losses in treatment 7 were also greater than losses observed at! lower measured oxygen diffusion rate and lower moisture content in treatment 11. These trends indicate that treatment 7 may have provided the microbiol population with an oxygen supply similar in some respects to the restricted supply found in treatments 11 and 12. In treatment 11, oxygen available at sites of microbial activity was restricted by reason of the small pores available for oxygen diffusion, since this fraction contained all the aggregates and fine particles less than . 17 mm. In treatments 7 and 9, diffusion of oxygen was measurably higher because of the larger pore spaces resulting from larger and more uniform aggregate sizes. At the low moisture tension (10 cm) involved in treatment 7, the effective availability of oxygen to microbial processes may have been restricted by thicker water films and by the presence of 33 locally anaerobic conditions within the saturated aggregates, giving rise to a pattern of nitrogen loss in treatment 7 that was essentially similar to that in the smallest aggregate size at a high moisture tension (Treatment 11). The higher moisture tension (35 cm) in treatment 9, together with the smaller size of aggregates involved would have reduced the gradient from the interior of the aggregate to the surface of moisture films in pores where the oxygen diffusion rate was the same as in treatment 7. This would have promoted greater uniformity in oxygen supply to all potential microhabitats in the liquid and solid phases of treatment 9 than of treatment 7.2 Some such relationship between moisture tension and aggregate size would appear to have operated to give rise to smaller losses of inorganic nitrogen in treatment 9 than in any other amended treatment, including those of larger aggregate size and higher measured oxygen diffusion rate. High Diffusion Rates Treatments 1 to 6 consisted of larger aggregates than those used in treatment 7, but all were adjusted to the same moisture tension (10 cm). As aggregate size increased from treatments 5 and 6 to treat- ments 1 and 2, moisture content decreased. Presumably the average thickness of moisture films would also have decreased with the decrease in number of aggregates and in the number of menisci associated with points of contact between aggregates. Significantly higher oxygen Zmin'l) were obtained with diffusion values (above 35 x 10'8 gms cm' increasing aggregate size in these treatments during the first six weeks of incubation (Tables 2 and 3). Thus, it may be visualized that these treatments represent a progressive increase in effective availability of oxygen to sites of microbial activity on the surfaces of soil aggregates. However, with increasing aggregate size there would have been an increased opportunity for interaction between aerobic surface habitats and anaerobic habitats within the interior of large aggregates. 34 With these considerations in mind, it will be observed (Figure 3—c) that nitrogen deficits in treatment 5 were much less extensive than in treatment 7, and were subject to much less extreme fluctuations. This response was associated with increased oxygen diffusion rate and with decreased thickness of water films. The behavior of the two largest aggregate sizes was distinctly different from other treatments in that extensive net mineralization of nitrogen occurred during the period from 35 to 43 days. These increases in mineral nitrogen were much greater in amended than in unamended soil. Losses of mineral nitrogen occurred, however, during the latter half of the incubation period, and these exhibited a distinctly cyclic pattern similar to those observed at lower oxygen diffusion rates (e.g. Treatments 7, ll, 13 and 15). Mineralization of organic nitrogen in soil is essentially an aerobic process involving respiratory loss of carbon and release of nitrogen as ammonium by deamination (Thimann, pp. 273-314; Waksman, pp. 480-503). Deamination can occur under anaerobic conditions, but at a much slower rate than under aerobic conditions (Waksman, 496, 499-500). The temporary large accumulations of mineral nitrogen, primarily ammonium, in the two largest aggregates reflects the aerobic nature of surface environments in these soils. However, the interior of these same aggregates are assumed to be saturated and will provide a locally anaerobic habitat for microbial populations. The alternate disappearance and reappearance of mineral nitrogen in the two largest aggregates at later stages of incubation would appear to reflect an interplay between these anaerobic p0pulations within the large aggregates and aerobic populations on their surfaces. The question arises as to whether inorganic nitrogen losses observed in the various treatments studied, were due to immobilization in organic forms or to denitrification. The limited data taken at the end of the experiment (Tables 10 and 11) show significantly higher values for organic nitrogen in the amended samples than in the unamended samples butaieinCOnclusive because no values for organic nitrogen were obtained at the beginning of the experiment. The differences observed could easily be accounted for either by a decrease in total organic nitrogen in the unamended samples or by an increase in the amended treatments. There would have to be an increase in total (organic and inorganic) nitrogen of approximately 100 to 2.50 PPM nitrogen for the latter to be true. This might be conceivable through fixation of atmos- pheric nitrogen, but it seems more probable that the observed difference is a product of both decreasing organic nitrogen levels in the unamended soils and increasing levels in the amended treatments. The only evidence in the collected data to suggest that denitrification occurred is the fact that, in water-logged soils and in soils with low diffusion rates, nitrogen appeared to be lost primarily at the expense of nitrate (Treatments 11, 13 and 15). Jansson and Clark (19) observed increasing nitrogen loss with increasing water content. In other treat- ments nitrogen deficits were accompanied by fluctuations in both nitrate and ammonium so that, conceivably, some reduction of nitrate to gaseous products may have occurred. Where denitrification occurs, it must start with nitrate or nitrite as the original substrate. It is essentially an anaerobic process in which nitrate is used as an oxygen source by microorganisms with the approPriate reducing enzymes (Thimann, pp. 359-368), but is not dependent on a completely anaerobic environment. However, biological reduction of nitrate under anaerobic conditions does not necessarily result in the evolution of gaseous products which are lost by volatili- zation (Waksman 542-557). Under strongly anaerobic conditions, nitrate can be reduced directly to ammonium. 36 Broadbent and ‘Stqjanovic (4) showed that with oxygen concen- trations of O. 1 percent and with 100 PPM tagged KNO3-N added, 27 percent of the added nitrogen was immobilized 2 percent was reduced to NH4-N, 1 percent was recovered as NO3-N and 70 percent was lost from the system: < At 1. 8 percent oxygen concentration the values were 56, 1, 8 and 35 percent, reSpectively. Because their work indi- cates that large quantities of nitrate are lost from soils underconditions of oxygen stress, we cannot exclude the possibility that loss of nitrate to gaseous ferms may constitute a substantial portion of the decreases in total mineral nitrogen observed in this study. The above workers also state that there was little evidence of nitrogen loss when nitrogen was added as ammonium salts. However, their experiment is of in- sufficient duration to prove that the nitrate, from the nitrification of the added ammonium salts, will not be denitrified. It is certain that denitrification and immobilization by reduction through assimilation can occur under both aerobic and anaerobic conditions. Accumulations of nitrite have been shown to accelerate nitrogen loss through denitrification by Fraps and Sterges (14) and Soulides and Clark (39). The soil reactions of amended treatments in the present study were below pH 6. O and ranged to a pH of 5. 2 and would not have inhibited the activity of Nitrobacter as has been reported to occur at neutral or alkaline reactions by Soulides and Clark (39), Aleem, e_t a_l. (1) and Chapman, e_t a_.l. (8). Fraps and Ste rges (14) also showed that nitrite was not found when ammonium sulfate was used as the source of ammonium until calcium carbonate was added. Periodic tests for nitrite in the treatments with low diffusion rates showed that nitrite was not present in more than trace quantities, as previOusly discussed. It seems reasonable to as surne that the transformation of ammonium to nitrate was not blocked at the nitrite stage, and that losses of gaseous nitrogen via nitrite were small if not negligible. 37 The fact that sufficient oxygen was available for extensive nitrate accumulation in all but the water-logged soils indicates that conditions were not strongly conducive to denitrification. The low pH of these soils and the absence of large quantities (of readily available energy mate rials in the form of added plant residues would also have tended to curtail denitrification (19). It is possible that chemical fixation of ammonium by soil organic materials may have occurred, but this is an oxidative process and is retarded by acid conditions (37). Since all of the treatments imposed conditions of oxygen stress on all or a major portion of the solid phase of these soil systems, and pH's were well below neutral, it is unlikely that appreciable chemical fixation occurred during the course of the incubation. However, Stojanovic and Broadbent (40) as well as Hiltbold gt 5:1. (17) were unable to quantitatively recover added ammonium from soils upon analysis before incubation. They suggested that chemi- cal fixation may be responsible for the low recpvery rates. Similar observations can be made from the data in this study, so some chemical fixation may have occurred in these soils initially. Considerable evidence is available to indicate that the primary factor involved in the disappearance of mineral nitrogen in this study was immobilization. Denitrification probably occurred to a slight extent in all the treatments and perhaps to a considerable extent in some of the saturated treatments with low oxygen diffusion rates, but appears to have generally played a minor role in inorganic nitrogen loss. Synchronous Fluctuations of Ammonium and Nitrate Unamended Samples The graphs in Figure 5 illustrate the cyclic fluctuations in ammonium and nitrate nitrogen which occurred in the unamended samples. PPM N T reatrnent 2 100- 4-6 mm aggreg. 10 cm HOH 50,. < 1 0 r T j— ! fi 10 20 3O 4O 50 60 Days of Incubation Treatment 4 100- 2-4 mm aggreg. 10 cm HOH 50$ 4 O 1 r I v I I 10 20 3O 4O 50 60 Days of Incubation Treatment 6 10 1-2 mm aggreg 10 cm HOH 1o 20 3b 46 so 60 Days of Incubation Treatment 8 10 .42-1.0 mm aggreg. 10 cm HOH Treatment 10 . .17-.4Z mm agg. 35 cm HOH W 7* 50 4b 50 60 f 10 26 Days of Incubation Treatment 12 é .17 mm aggreg. 400 cm HOH l 1b 50 3b 46 5b 60 Days of Incubation Treatment 14 - .17-.42 mm aggreg. Saturated m 1b 26 30 40 Days of Incubation Treatment 16 é. 17 mm aggreg. Saturated i6 253 3d 45 56 66 Days of Incubation Figure 5. samples with time. 7b b 1b 20 30 4O 50 6O 70 Days of Incubation Ammonium and nitrate nitrogen appearing in the unamended 39 There was an increase in the amount of organic nitrogen mineralized (ammonium plus nitrate) as the aggregate size decreased from treat- ment 2 where little net mineralization occurred to treatment 10 where the greatest amount of mineralization occurred and abrupt changes in nitrate levels were apparent. In the two largest aggregate sizes (Figure 5, Treatments 2 and 4) nitrate accumulation exhibited a pattern more or less characteristic for the normal bacterial growth curve, with a lag phase, a maximum rate phase and a climax or leveling off. Synchronous fluctuations of ammonium and nitrate disrupted this characteristic appearance in other treatments. These fluctuations became progressively more violent with decreasing oxygen diffusion rate, and consequently with decreasing aggregate size, until saturation was reached. Periods of ammonium accumulation occurred approxi~ mately seven days after periods of nitrate accumulation. These sequences indicate that ammonifying populations became increasingly dependent upon nitrate as a source of combined oxygen as their environment became increasingly anaerobic. The saturated soils showed loss of nitrate early in the incubation with only slight, transient accumulations after 35 days, and fluctuations in ammonium were accordingly subdued after the initial rapid increase. There was no direct equivalence in the reciprocal increases and decreases in ammonium and nitrate. Large increases in nitrate rarely resulted in equally large decreases in ammonium, indicating that ammonium was being more or less continuously released by decomposi- tion of organic materials. However, ammonification did not proceed at a uniform rate. Successive peaks in the ammonium curve represent periods of enhanced activity associated with changes in the heterotrOphic population. These ammonium peaks coincided with disproportionately large decreases in nitrate. 40 This decrease in nitrate may have resulted from immobilization or denitrification. ' Nitrate may have served as a terminal hydrogen acceptor in the reapiration of the heterotrophic ammonifying population. Gaseous products may have been produced from this reduction process, but at least a portion of the nitrogen could have been assimilated by the active ammonifying pepulation. ‘ The moderate pH and lack of fresh organic matter were not specifically conducive to denitrification. Nitrifying organisms may have been inhibited by soluble fermentation products diffusing from anaerobic sites within the aggregates. Amended Samples The data from amnended samples are graphed in a manner similar to the unamended samples (Figures 6 to 9). In some instances the curves are similar to those of the corresponding unamended samples, but in others they are quite different. . Ammonification occurring early in the incubation in the amended samples was greater in the larger aggregate sizes and diminished as the aggregate size decreased. No mineralization occurred in treatment 11. -Also, in sharp contrast to the unamended series, fluctuations in ammonium levels were much more extreme in the better aerated soils, and became less and less violent with decreas- ing aggregate size and decreasing oxygen diffusion rate. The beginning of the maximum rate phase of nitrification was generally less distinct in the amended soils than it was in the unamended soils because measured nitrate levels fluctuated erratically, making it impossible to define a satisfactory curve (NO3-N curve in Figures 6 to 8). Dips in this nitrate accumulation curve did tend to coincide with or slightly precede periods of ammonium accumulation, as was true in the unamended soils. The opposing tendencies toward more extreme ammonium fluctu- ations in larger aggregates with added ammonium sulfate and in smaller aggregates in unamended soils are related to the relative quantities of 41 350 ” NH INVERT. ' 300 ~ 4 250 _ I z I MINERTAL N O z 200 t I; L S a TREATMENTI I ‘ N0 _~ °' I 50 . 4-6 mm AGGREGATES I 53 , 3 IOcm wuss TENSION! m _, I I '00 " .’ M, NH4'N I . 50 L ' [I T 1 1 1 i 4 L :rm 1 1 l I 0 IO 20 30 4O 50 60 DAYS OF INCUBATION 350’ NH4INVERT. 300* 250 MINERAL z N LOST 2 200- a TREATMENT 3 N0 -N 0- | 50- 2-4mm AGGREGATES ' 3 lOcm WATER TENSION NH4_N I 00 - :30L -——"""“‘\x O 0 0-4 | L 1 J 4 4 1 A 0 I0 20 30 4o 50 so DAYS OF INCUBATION ‘ Figure 6, Ammonium and nitrate nitrogen that occurred in the amended samples, treatments 1 and 3, with time. The arrow joining the horizontal axis indicate lag time (t'). 42 350T 300 250- NH4INVI'RT. TREATMENT 5 7' z ZOOF I-2 mm AGGREGATES MWERAL z . IO Cm WATER TENSION / \\\§‘i N LOST a. ‘ ' i--'-.‘f-s-.-.- “ < H .. IQOt 4: N 4 N 50 ” . 1.1 I I L 1 l l 1 l A L. 0 IO 20 . 30 4O 50 60 DAYS OF INCUBATION 35° H4INVERT. 300L ‘ 250 t 2 200“ TREATMENT 7 MINERAL 3 .42-Imm AGGREGATES \\\\ \\.,\\\ Tit-(‘3'; N LOST & '50T IO cm WATER Tsusuou '- ‘ '00 b . NO3-N 501- NH4-N 0 IO 20 DAYS OF INCUBATION Figure 7. Nitrification patterns in the amended soils, treatments 5 and 7. The NH4 invert curve is used as an estirnate of nitrification. Arrows joining the horizontal axis indicate lag time (t‘). 43 350 300 250 _ NH4 INVERT. Z 200— TREATMENT 9 . I WNERAL 2 .u7-.42 mm AGGREGATES ,’ N LOST CL 35 cm WATER TENSION Q N03'N a. '50 f" ‘ NH4-N I00} I 50 #A\_ I 1 l __1 0 IO 20 3O 4O 50 60 DAYS OF INCUBATION 350 ' 300 250 . 2 200 ” TREATMENT II 5 , 2: "\' :VLRCK: . §\\ 7 < 33: \\ \ \3\\\\\ \‘h \\ s _. “a“ §\ “§ N L o s r . \\ ix? .x\\“\ .\\“ . x \ i \ _. . .._K. 2?‘\., \\ \ ‘9‘,“ .T J“ <2‘-.;\\\‘-Z\§ . «. \\\\\\ ‘2 1:. \~ -.‘\\\.A:\\:\>‘ ~ -;: .. ‘0 QQ‘ ‘\'x -\ ‘-\ ‘\‘\‘-' :Wm \\\\-\\ ‘1. \ {:\ \. x- .\:5‘3?\:\ .-.. \\.\‘ ' ~{\‘r‘ ; ‘ \». *\\\ K\\\\§\:\\\ \ ,\ »\\ Q” \gk‘ . E .»\ .x‘ ‘ . ‘ \ V _\ - ‘ m- \‘\ ~ _, \\ \‘Xfig ‘ix \ ‘ “ ‘ ' t 2‘ \ \ \-.. ._‘-, \ ‘\\ c _\O.‘. Q.“ ~\- . \* ~ .- \\ ‘\\\\\\\"‘.C\\\ \ .—_ . 50 o L..__-, w. -1 . <. \\ IO 20 30 DAYS OF INCUBATION ' NO3-N TREATMENT 15 <.I7mm AGGREGATES NH INVERT. r SATURATED "4 NH4'N s , MINERAL ’ ‘ N LOST . '. .- .xgss} . . \\\\\\\\x\\ go; ,. ' \\ .‘ .\\ , . "03-“. IO 20 M 30 40 so so DAYS or INCUBATION Figure 9. Nitrification patterns in the amended soils, treatments 13 and 15, with time. The NH, invert curve is used as an estimate of nitrification. Arrows joining the horizontal axis indicate lag time (t'). 45 sulfate and nitrate present to supply combined oxygen for anaerobic processes. ~ Thus, the large quantities of sulfate added to the amended soils provided a ready supply of combined oxygen in soluble form to support respiration linked with sulfate reduction. In the larger separates, this type of reSpiration would have been favored within the aggregates. A gradient of decreasing dependence on sulfate can be visualized, proceeding from the interior of the aggregates toward more normal aerobic reSpiration at the surface of moisture films surrounding the aggregates. With decreasing aggregate size (or with decreased thickness of moisture films as in the case of treatments 9 and 11), the gradient between conditions favoring these two types of me tabolism would have been reduced, providing less and less opportunity for interactions between them. In the unamended soils, the principal combined oxygen source at the beginning of the experiment would have been the nitrate initially present. The quantities initially present were relatively low and were rather quickly depleted by assimilation or reduction to ammonium at low levels of oxygen availability (Figure 5, Treatments 6, 8 and 12). It was only after active nitrification had begun that periodic reduction of nitrate to support heterotrophic respiration became a dominant feature in all except the two largest aggregates and the two saturated soils. The observed fluctuations in ammonium and nitrate may be con- sidered to represent an interplay between anaerobic processes linked with reduction of sulfate or nitrate and aerobic processes which would include nitrification and oxidative ammonification. The reduction of nitrate was not essentially a direct conversion of nitrate to ammonium because ammonium .did not appear in quantities equivalent to the quantities of nitrate lost. Broadbent and Stojanovic (4) observed that little added nitrate is reduced to ammonium under conditions of oxygen stress in the presence of added organic matter, as mentioned previously. 46 In unamended soils, periodic accumulations of ammonium were much smaller than the corresponding losses of nitrate (Figure 5). In the soils to which ammonium sulfate was added (Figures 6 to 9), periodic peaks of ammonium accumulation far exceeded the magnitude of corresw ponding dips in the nitrate accumulation curves. Denitrification would not appear to have been a major factor in nitrate disappearance in these soil systems, with the possible exception of the saturated small aggregates (see previous section). Thus, (nitrate reduction would appear to have been predominately assimilatory, the necessary energy having been supplied from organic substrates by heterotrophic organisms which made use of nitrate as a source of nitrogen as well as of combined oxygen in the absence of adequate supplies of molecular oxygen. In the unamended soils, the carbon-nitrogen ratio of organic subu- strates was apparently favorable for retention in microbial cells of a large part of the reduced nitrogen from nitrate. As a result, only small amounts of ammonium were released, and fluctuations in ammonium we re much less extreme than for nitrate (Figure 4). In contrast to this, early adjustments in the presence of excess ammonium nitrogen in amended soils would have given rise to organic substrates rich in nitrogen. Decomposition of these by heterotrophes growing anaerobically by preferential assimilation of nitrate arising from nitrification would have resulted in ammonification of a portion of the contained nitrogen. Net ammonification would have been enhanced where diffusion of soluble fermentation products to more aerobic habitats near the surface of water films could occur, giving rise to more extensive respiratory losses of carbonand more extensive breakdown of nitro— genous organic compounds. Thus, reduction of relatively small amounts of nitrate would have resulted during the disproportionately large accumulations of ammonium observed in amended soils, and particularly in the larger aggregate sizes (Figures 6 to 9). 47 Estimation of Nitrification Rates and Lag Times The first attempt at determining maximum rates (K values) and lag times (t') for each of the amended samples, was made by drawing a smooth curve through the points of measured nitrate nitrogen. The difficulty encountered can be observed in the graph of nitrate measured in treatment 11, figure 8. Drawing a smooth curve through such erratic points, is difficult. Any resulting curve would not be a reliable one, nor would it display the characteristics of microbial growth. The ammonium points on the same graph, however, form a curve which is ideally representative of microbial growth and activity. The maxi- mum rate phase of the ammonium disappearance curve is linear and uninterrupted until the growth and activity rate of the. nitrifying organisms decreases. This would tend to indicate that ammonium disappearance resulted from consumption only by nitrifying organisms and that nitrogen was not being assimilated in the ammonium form by other groups. By adding nitrogen deficit values from the graph for this treatment in figure 4 to the measured nitrate values, we can plot an inverted image of the ammonium curve and obtain estimates of both maximum rate and lag time that should represent produced nitrate. The use of the inverted ammonium curve to represent nitrifi- cation in the soil systems studied here involves several assumptions based on material which has previously been discussed. These are summarized as follows: 1. Nitrate nitrogen has been assimilated preferentially over ammonium as a source of nitrogen by heterotrophic organisms, in treatments where molecular oxygen is present in limited quantities, because of low measured oxygen diffusion rates. 2. Interaction between aerobic and anaerobic environments has occurred in treatments involving higher diffusion rates and 48 large aggregate sizes. Nitrate has been preferentially assimilated as a source of oxygen and nitrogen in the locally anaerobic microhabitats of these treatments. 3. Changes in ammonium nitrogen levels reflect nitrification which has occurred in all treatments as it appears to reflect nitrification in treatment 11. 4. Nitrite nitrogen did not accumulate to any considerable extent to facilitate denitrification or to interrupt the nitrification cycle. The graph of treatment 7 in figure 7 shows a secondary minerali— zation cycle in the ammonium curve beginning after 38 days incubation which, when inverted, gives the impression that the linear phase of the maximum rate is discontinuous. A very significant feature of this adjusted nitrate curve is the close similarity of the slope in successive sectors of accumulation. A continuous maximum rate phase can be obtained, for this and similar treatments with multicyclic ammonifi- cation curves, by assuming that nitrification rates continue at a maximum rate, as seems to be indicated, once they are initiated. The secondary maximum rate phase appearing in the inverted curve for treatment 7 beginning at 48 days can be moved horizontally across the graph until it intersects the linear phase of the earlier maximum rate, as indicated by the arrows, to give a composite maximum rate line. Composite lines prepared in this way for each treatment were used to estimate the K values shown on each of the graphs in figures 6 to 9. The nitrifi- cation rates (K values) obtained by this procedure appear to be valid and are well within the range of values reported by other workers (34). Another procedure could be used in displacing the maximum rate phases of the secondary mineralization cycles. .They could be moved diagonally to the upper left with the vertical component corresponding 49 to the increase in ammonium nitrogen observed in the bar graph of treatment 7 in figure 3d and the horizontal component of sufficient magnitude to make the composite maximum rate curve continuously linear. Either method indicates the same slope in the maximum rate phase, 80, it is not overly important which method is employed. The major difference is that the position of the diminishing activity phase will be at a higher level of nitrate nitrogen with the second method. Since there is insufficient data to say that some of the treatments have reached the point of diminishing activity except in the case of treatment 11, the second method would not be any more reveal- ing than the first. Lag times were difficult to estimate with any assurance because of the uncertainty as to what nitrate value should be used as a base line. The t' values shown in figures 6 to 9 were derived by extending the maximum rate line to the point of its intersection with a base line taken as the lowest nitrate value observed during the lag period. . A further difficulty arose in the case of the two largest aggregates (Treatments 1 and 3). Here it was impossible to establish the exact time when a maximum rate phase was actually initiated. In treatment 1 it appeared that nitrification had commenced by the 28th day, but nitrate was utilized as rapidly as it was formed to support respiration and growth of the ammonifying populations responsible for the large peak in ammonium levels apparent during the following two weeks. The lag time based on this transient nitrate peak appears more reasonable than that based on the later more sustained cycle. In treatment 3, reduction of nitrate to support growth of ammonifying organisms appears to have completely obscured the actual beginning 3of nitrification, and the first net accumulation of nitrate probably occurred well after a maximum rate phase of nitrification had been established. 50 Adjusted nitrate curves for saturated soils gave some indication that nitrification may have occurred to a limited extent. It is quite likely that this occurred in the surface layers of the soil columns and, that, in the main body of the soil, nitrification was indefinitely retarded. Lag Time and Oxygen Diffusion Rates The observed relationships between lag time (t') and oxygen diffusion rates in amended soils are shown graphically in figure 10. The t' values were calculated as described above and plotted against the average oxygen diffusion rate for the entire lag period. Except for the two saturated soils in which nitrification was indefinitely retarded, lag times increased with increasing oxygen diffusion rate. Since nitrification is an aerobic process, this is contrary to expectation. However, the increasing lag times were directly related to inc reas— ing activity of ammonia producing organisms (Figures 6 to 9). In the smallest aggregate size (Treatment 11), there was no evidence of net accumulations of ammonium. at any time. With increasing oxygen availability, peaks of ammonium accumulation increased in frequency and magnitude. As noted in the previous section, assimilatory reduction of nitrate during growth of ammonifying organisms under anaerobic or mildly anaerobic conditions resulted, apparently, in the utilization of nitrate as rapidly as it was formed during the first stages of rapid nitrification. This obviously obscured the end of the actual lag period in treatments 1 and 3. A similar effect was probably expressed in diminishing degree as ammonifying activity decreased with decreasing oxygen diffusion rate. Thus, the net effect of increasing oxygen diffusion rate was to delay the equilibrium accumulation of nitrate rather than the process of nitrification itself. LAG TIME DAYS 01 A U 0 OJ 0 25t 1 1 To 20 so 440 So 20 OXYGEN DIFFUSION RATE -gm oz x I0’° cm"2 min." Figure 10. Lag times (t') as they were affected by oxygen diffusion rates. 52 Maximum Rate of Nitrification and Oxygen Diffusion Rate Maximum nitrification rates (K values) are plotted against the average oxygen diffusion rates for the incubation period following establishment of maximum rates in figure 11. There was a gradual increase in K values as oxygen diffusion rates increased from about 1 “'2 min” . 5 to about 35 x 10'"8 gms cm There was an abrupt increase in K values at slightly higher oxygen diffusion rates in the two largest aggregates (Treatments 1 and 3). The diffusion rates in treatments 1 and 3 were only occasionally significantly higher than in treatment 5 (Table 2). So, it appears that some factor other than oxygen diffusionwas responsible for the sharp increase in calculated K values for treatments 1 and 3. All K values were calculated on the basis of the adjusted nitrate curves in figures 6 to 9. This adjustment was based on the premise that the only way in which mineral nitrogen was lost from these systems was by the preferential assimilation of nitrate by heterotropic organisms. This assumption would appear to be valid under conditions of oxygen stress where nitrate would be utilized both as a source of oxygen for respiration and as a source of nitrogen for synthesis of cell proteins. However, it is known that under aerobic conditions many soil organisms preferentially assimilate ammonium when both ammonium and nitrate are available. - Since the calculated K values. reflect all losses of mineral nitrogen, the high values for treatments 1 and 3 most probably reflect a sharply increased assimilation of ammonium in addition to nitrate. The validity of these two values is, therefore, highly questionable. The K values for other treatments are subject to a similar criticism. However, the close similarity in slope of successive segments of the calculated curve for nitrate accumulation and ammonium disappearance (Figures 6 to 9) suggests strongly that but one continuing process was 53 4oo~ X DJ E” \ 300L 2 I IO 0 Z 5 200; Q. Q. I U) ‘5’ _IIooL < > x O l l L 9 IO 20 so 40 ,so OXYGEN DIFFUSION RATE -9m 02 x I0‘° cm"2 min." Figure 11. Maximum nitrification rates as they were affected by oxygen diffusion rates. 54 involved. Microbial growth and assimilation are highly cyclic phenomena. It is very unlikely that cycles of ammonium assimilation would be so closely synchronized with cycles of ammonium oxidation (or nitrification). Furthermore, no cyclic pattern of nitrification would be expected as long as unlimiting supplies of ammonium were present. At no time could ammonium be considered limiting in these amended soils. By process of elimination, nitrification remains as the one continuing process that can most likely account for the successive periods of uniform ammonium disappearance. The maximum nitrification rates obtained indicate that the nitri- fication process wa s not dependent on the surface area of soil aggregates. The surface area of aggregates contained in a unit volume of soil will increase as the aggregate size decreases giving an inverse relationship to the maximum rates obtained in this study. Threshold and Critical Levels of Oxygen Diffusion The [data in figure 11 suggest that oxygen diffusion rate became absolutely limiting for nitrification somewhere in the range of 8 to l 2 min" . 10 x 10'8 gm cm‘ These data'represented average diffusion rates for major portions of the incubation period. A second basis for evaluation of threshold levels of oxygen diffusion was obtained by plotting nitrate measured against oxygen diffusion rate for individual replicates for each sampling date. This relationship for determinations made 40 to 43 days after the beginning of incubation is shown in figure 12. The plotted values represent a time when the maximum rate phase of nitrification had definitely been established in all but the saturated soils. No oxygen diffusion rate appears to be absolutely limiting in this graph because of some carry over of nitrate from that originally present in the. soils NITRATE NITROGEN PP M ISO I40 I30 IZO IIO I00 55 fl 0 AMENDED SAMPLES OUNAMENDED SAMPLES I ”T L. I I— so, 80 f T O IO 20 so 40 so OXYGEN DIFFUSION RATE -qm 02 x IO"8 cm"2 min.‘! Figure 12.. The relation of nitrate nitrogen accumulation to oxygen after 40 to 43 days of incubation. 56 of some treatments. However, as oxygen diffusion rate increased I 2 min" , accumulated nitrate from the range of l to 10 x 10'8 gms cm“ levels increased in an apparently hyperbolic manner, tending to level 1 7‘ min' . off above 30 x 10'8 gms cm‘ Finn (13) has reported similar responses in microbial respiration rates occurring below a critical oxygen level, which he refers to as the point below which oxygen concentration begins to effect respiration rate. Broadbent (5) has also shown a similar response in nitrification rates using partial pressures of oxygen to limit oxygen supply. At later sampling dates, an absolute threshold level of oxygen diffusion became apparent, below which no nitrification occurred. The data for 49 to 50 days incubation (Figure 13) indicates that this was I 2 min“ . Maximum accumulations in the range of 3 to 10 x 10“8 gms cm- 2 1 of nitrate occurred at about 30 x 10'8 gms cm‘ min" , the level which appeared to be critical in the earlier sampling period. However, the range of observed values was greatly increased, reflecting the dis- ruptive effect on nitrate accumulation of cyclic mineralization and assimilation of nitrogen. The sharp drop in maximum nitrate accumulation above 30 x Z 10"8 gms cm' min"1 indicates that the extent of assimilative immobili- zation of nitrogen was enhanced at higher oxygen diffusion rates. 2 I It thus becomes difficult to say whether 30 x 10"3 gms cm' min‘ represents a critical level above which nitrification er s_e was no longer dependent upon oxygen availability, or whether this represents an optimum balance between heterotrophic and autotrophic requirements. Below this level, however, maximum nitrate accumulations were definitely a function of oxygen diffusion rate until the absolute minimum -2 1 threshold level was reached. Therefore, 30 x 10‘8 gms cm min' may represent both a critical and an optimum oxygen diffusion rate for maintaining high nitrate levels in the soil systems studied. 57 I60 1 0 AMENDED SAMPLES OUNAMENDED SAMPLES 1 I50 I40 T I30t I20 I I ”of IOOL 80" NITRATE NITROGEN P P M \I _<2___ O IO £0 £0 40 so OXYGEN DIFFUSION RATE -qm 02 x Io-B cm-z mln." Figure 13. Relation of nitrate nitrogen accumulation to oxygen diffusion rate at 49 to 50 days of incubation. 58 Similar critical oxygen diffusion rates measured with the platinum microelectrode were reported by Van Doren (45) for sugar beets, tomatoes, oats, corn, and potatoes which ranged from 25 to 40 x 10“8 gms cm"z min‘l. Practical Implications The results obtained by other workers using partial pressures of oxygen do not 'contradict findings in this study employing diffusion rates. Controlling partial pressures of oxygen in the soil atmOSphere in effect limits the diffusion of oxygen through water films, soil aggregates and cell walls so that similar responses should be expected. Partial pressures, however, can only be used in the laboratory to define the relationship that exists between nitrification and oxygen supply but do ' not facilitate the prediction of nitrification in field situations as affected by aeration. Partial pressure methods also have the disadvantage that the amount of oxygen available to an organism may vary as soil con- ditions suchas water content, aggregate size and pore distribution vary between soils. ~ If the substrate through which oxygen must pass to reach microorganism inhibits gaseous diffusion, the relationship can only be evaluated by measuring diffusion rate. _ Measuring oxygen diffusion with a platinum electrode in situ provides aerationdata that could be an important step in making quanti- tative predictions of this nature. Sabey, e_t a-._l. (35) presented a method whichvmay show promise in making quantitative predictions of nitrification rates for soils if aeration‘and other environmental factors could be properly evaluated. 11: is interesting to note that nitrification was inhibited only in the saturated treatments with some exceptions occurring in occasional other tubes with very low diffusion rates. Nitrification appeared at 59 diffusion rates only slightly above those Observed in the saturated samples. The conclusions of early workers such as Schloessing and Muntz, Gainey and Metzler and others that oxygen concentrations rarely occur in field conditions at levels which have been demonstrated to inhibit nitrification in the laboratory, may be quite correct, without consideration for nitrification rate. However, we might add that high diffusion rates gave greater maximum rates than lower diffusion rates, in this study, and even though this was not inhibition, it .most certainly was affecting nitrification in a very substantial way. Theron (42) stated that plants Stipress nitrification and proposed that roots have a bacteriostatic effect on organisms. He also states that ammonium appeared in more than normal quantities while nitrifi~ cation was depressed. He concluded that the plant exerts its influence on the autotrophic dehydrogenase system of the nitrifiers and not on the process of ammonification. Lyon e_t a_._l. (25) suggested that carbon.» aceous material given off by roots may favor the development of nitrate consuming organisms in the soil with the consequent transformation of nitrate into other nitrogenous forms. The assimilation of nitrate nitrogen by mineralizers or possible nitrate loss through denitrification, resulting from an active microbial population, can account for the low apparent rates of nitrification observed under grass by the above workers. It seems reasonable to exPect that there would be a high nitrate require- ment among both the mineralizing organisms and the grass vegetation and that nitrate would not accumulate in quantities comparable to ammonium as it would be continually assimilated by the other organisms. If the assumptions in this study are correct and the factors involved comparable, then the data of this study does not support the conclusion of Theron. 6O Criticism and Evaluation of Methods The objectives of this study outlined at the beginning were not achieved using the data in its original form. Lag times (t') and maximum nitrification rates (K values) should have been obtained without having to adjust’for mineralization cycles and mineral nitrogen losses if all environmental factors had been adequately controlled. Untreated samples were originallyincluded in the incubation so that nitrification rates could have been computed on the basis of added ammonium nitrified. However, lack of any synchronization between amended and unamended treatments made nitrate values, calculated as percent of added ammonium, completely meaningless. It was found that several factors contributed to the cyclic fluctu- ations of ammonium and nitrate in most samples and necessitated a hypothetical rather than a statistical solution for the responses observed. These fluctuations could have been diminished by altering some of the materials and methods used. These factors and possible solutions are summarized as follows: 1. The soil had been stored air-dry for a period of four years, causing the beginning microbial population to be quite small. It was hoped that this condition would accentuate the differences in rate of proliferation between treatments. It was found that the interplay between microbial populations indicated by multicyclic mineralization. and nitrate immobilization was an over riding disadvantage. This difficulty could have been reduced by preincubating the soil as has been done by Sabey, e_t a}. (34), and also by obtaining an original sample that had recently been procured from a field site. 2. Oxygen diffusion rates were controlled and an effective dif- fusion range obtained by varying moisture levels and aggregate 61 size. In the larger aggregate separates, aerobic micro- habitats existed on the surface of aggregates and increasingly anaerobic microhabitats existed within the aggregates as the distance from the surface increased. The resulting interplay between surface aerobic populations and subsurface anaerobic pOpulations appeared to be the largest single factor causing multicyclic fluctuations in ammonium and nitrate. This problem appears to be difficult to overcome because other methods of controlling diffusion rate by adjusting soil physical conditions are not obvious at this time. The interactions could probably be decreased by using aggregate separates that are less than 2 mm in diameter along with moisture levels between about 20 and 400 cm tension, thus obtaining an effective diffusion range with smaller aggregate separates and a wider range of moisture content. Other workers (Fraps and Sterges, Greaves and Carter) have shown varying nitrification rates with changing moisture levels. It remains to be shown whether or not their reported reSponses are entirely a direct product of soil moisture or whether resulting aeration levels are responsible for some of the observed responses. .- Ammonium sulfate was used as a source of ammonium in the amended treatments. Much greater mineralization rates occurred in the large amended aggregates than occurred in unamended aggregates of the same size. The presence of sulfate as well as nitrate provided a source of combined oxygen to microorganisms in anaerobic microhabitats and accelerated mineralization at these sites. Perhaps by using another ammonium source, this response could be decreased. However, there are advantages to using ammonium sulfate, 62 because several workers (Fraps and Sterges) have observed negligible nitrogen losses from soils amended with ammonium sulfate. Few other ammonium sources have this character.» istic. Chemical fixation of ammonium may have occurred extensively in most samples at the beginning of the incubation. Amended soils were treated with 400 PPM (NI-I4)ZSO4 and only 220 to 320 PPM were recovered in the initial analysis. Stojanovic and Broadbent (40) and Hiltbold et a}. (17) have reported similar difficulties and suggest that chemical fixation may be responsible. In this study the rate of recovery decreased with increasing aggregate size, indicating that some of the discrepancy is probably associated with the method of application employed since it was difficult to obtain uniform distribution of added ammonium in the larger aggregates. Additional total nitrogen determinations would naturally have facilitated a more complete and conclusive interpretation of the responses obtained. The few analyses made serve merely as an indication of the type of data that should be obtained in future studies of this nature. N15 studies would also provide significant evidence in determining what factors were responsible for nitrate loss. Conway's microdiffusion technique for determining ammonium and nitrate nitrogen developed for use with soil extracts by Bremner and ‘Shaw (3) provided reliable and reproducible data throughout the study. The procedure has many advantages in that only one extraction is necessary, colored filtrates do not interfere, and the nitrate determin- ation is only slightly different from the ammonium determination. They can therefore be run simultaneously. Measured oxygen diffusion rates were effectively controlled by aggregate size and moisture tension. However, altering these physical 63 dimensions also changed the ratio of micro-aerobic habitats to micro- anaerobic habitats from treatment to treatment and complicated the interpretation of observed responses. Even though aggregate size and moisture tension appear to be the only methods presently available for adjusting diffusion rates in soils by natural means, they are certainly not ideal for a study of relationships that exist between oxygen diffusion rate and nitrification. The platinum microelectrode used for oxygen diffusion measure- ments in this study apparently did not measure diffusion rates exactly as they affect the‘microenvironments in the soil. Locally anaerobic conditions existing in primarily aerobic treatments were not detected by diffusion measurements. SUMMARY AND CONCLUSIONS Oxygen diffusion rates were measured with the platinum mic row electrode in soil systems made up of varying size aggregate separates and moisture tensions. Nitrate and ammonium nitrogen were determined to estimate lag times and maximum nitrification rates as influenced by oxygen diffusion rates. Measured nitrate curves lacked patterns which were characteristic for nitrification in homogeneous soil systems. Heterotrophic organisms in locally anaerobic mic rohabitats within primarily aerobic treatments may have utilized combined oxygen from produced nitrate giving rise to reductive and assimilative losses of nitrate. Consequently, nitrate levels measured were equilibrium values of nitrate accumulation and did not reflect the actual conversion of ammonium to nitrate. The relationship between oxygen diffusion rates and nitrification was assessed on the basis of ammonium disappearance rather than nitrate appearance. The inverted ammonium curve gave a basis for estimating maximum nitrification rate and lag time. The values obtained are valid measures of nitrification if all ammonium disappearance is assumed to be a result of nitrification. There was some evidence in the larger aggregate separates that ammonium was being utilized by other processes. Lag time obtained increased‘with increasing diffusion rate and aggregate size except at the very low oxygen diffusion rates in saturated soils, where the maximum rate phase was never reached. This unexpected response appeared to be due to effects of moisture tension and aggregate size on the availability of oxygen to microbial populations within aggre- gates. Moisture levels used produced saturated conditions within the 64 65 larger soil aggregates. As aggregate size increased, the ratio of anaerobic to aerobic habitats became larger in spite of the fact that oxygen diffusion rates measured with the platinum electrode increased. Reduction or assimilation of nitrate by anaerobic pOpulations or inhibitory effects of soluble fermentation products on the nitrifiers, may have been responsible for delayed nitrification in these treatments. Maximum nitrification rates increased with increasing diffusion rate. The rate increased more sharply among the very large separates than it did among the treatments with lower diffusion rates. This suggests that ammonium may have been used in some other soil process in addition to nitrification and that the estimated K values for these highest diffusion rates were not completely valid. Relationships observed between oxygen diffusion rates and measured nitrate levels are summarized as follows: 1. A threshold level, the point below which nitrification did not occur, was found between 3 and 10 x 10'8 gms 02 cm”2 min”, The minimum value became more sharply defined as the experiment progressed with time. 2. During the period when most samples were in the early stages of the maximum rate phase, measured nitrate levels increased in a hyperbolic manner with increasing oxygen diffusion rate to 30 x 10"8 gms 02 cm"2 min‘l. Above this level nitrate accumulation was not influenced by oxygen diffusion rate. 3. Later in the maximum rate phase, a very rapid increase in nitrate levels was observed as the diffusion rate increased to 2 min"1 This was followed by a decrease 30 x 10"8 gms cm- in nitrate levels as the diffusion rate increased to 55 x 10"8 gms cm'z min" Increased activity of heterotrophic organisms and consequent assimilation of nitrate or antagonistic 66 suppression of nitrification may have caused this observed decrease in nitrate levels. The oxygen diffusion rate of 2 l 30 x 10‘8 gms cm“ min" may represent both an optimum and a critical level for the accumulation of nitrate in the presence of a mixed soil microbial population. Previous estimates of critical levels and threshold levels have been made by various workers using partial pressures of oxygen. . A comparison of these values with diffusion rate values found in this study follows: 1. A threshold level of 0. 2 to O. 4 precent 02 was reported by Amer and Bartholomew (2) for soils that they studied. A comparable value in terms of diffusion rate appears to be 1 “'2 min“ . between 3 to 10 x 10“8 gms 02 cm 2. Critical levels in terms of partial pressure for nitrification are reported to be around 11 percent by several workers (2, 36). A comparable diffusion rate for soils in this study would be 30 x 10""8 gms Oz cm"7‘ min‘l. 3. Oxygen concentrations below 11 percent have resulted in nitrate levels which decreased in an approximately hyperbolic manner (2). , A similar re8ponse was observed in this study ’2 min“l. below 30 x 10'"8 gms 02 cm 10. 11. BIBLIOGRAPHY .- Aleem, M. I. H., Engel, M. S., and Alexander, M. The inhibition of nitrification by ammonium. Bacterial. Proc. Soc. Am. Bacteriologists. .P. 9, 1957. . Amer, F. M. and Bartholomew, W. V. Influence of oxygen conceiv- tration in soil air on nitrification. Soil Sci. 71:215-219. 1951. . Bremmer, J. M. and Shaw, K. Determination of ammonium and nitrate in soil. Jour. of Agric. Sci. 46:320-328. 1955. . 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W. and Brink, F. Microelectrodes for measuring oxygen tension in animal tissues. Rev. Sci. Inst. 13:524-333. 1942. 67 12. 13. 14. 15. l6. 17. 18. 19. 20. 21. 22. 23. 68 . Duncan, D. B. Multiple range and multiple F tests. Biometrics II, 1-42. 1955. Finn, R. K. Agitation-aeration in the laboratory and in industry. Bact. Rev. 18:254-274. 1954. Fraps, G.-S. andSterges, A. J. Nitrification capacities of Texas soil types and factors which affect nitrification. Texas Agr. Exp. Sta. Bul. 693. 1947. Frederick, L. R. The formation of ammonium nitrogen in soils: I effect of temperature, Soil Sci. Soc. Amer. Proc. 20:496-500. 1956. Gainey, P. L., and Metzler, L. F. Some factors affecting nitrate nitrogen accumulation in soils. Jour. Agr. Res. 11:43-64. 1917. Hiltbold, A. E., Bartholomew, W. V. and Werkrnan, C. H. The use of tracer techniques in the simultaneous measurement of mineralization and immobilization of nitrogen in soil. Soil Sci. Soc. Amer. Proc. 15:166-173. 1951. Keen, B, A, The Physical Pr0perties of Soils (Chapter X, The soil n ”w- atmOSphere) pp. 334-354, LOngmans, tGrueen and Co., London. 1931. Jansson, S. L. and Clark, F. E. Losses of nitrogen during decomposition of plant material in the presence of inorganic nitrogen. Soil Sci.- Soc.-Amer. Proc. 16:330-334. 1952. Kolthoff, L. M., and Lingane, J. J. Polarography. Interscience Publishers, Inc. , New York (1946). Korsakova, M. ~ P. Effect of aeration on the process of bacterial nitrate reduction. Mikrobiol. (U.S.S. R.) 10:163-178. 1941. (Abstract in Chem. Abs. 36:4848). Laitinen, H. A., and Kolthoff, I. M. 'A study of diffusion processes by electrolysis with microelectrodes. J. Am. Chem. Soc. 61:3344-3349. 1939. Lemon, E. R. .5011 aeration and its characterization. Ph. D. Thesis. Michigan State College. 1952. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.. 69 Lemon, E- R. , and Erickson, A.- E. The measurement of oxygen diffusion in the soil with a platinum mic roelectrode. Soil Sci. Soc.~ Amer. Proc. 16:160-163. 1952. Lyon, T. L., Bissell, J.-A., and Wilson, B. D. Depressive influence of certain higher plants on the accumulation of nitrates in soil. Jour. Arner.- Soc.Agron, 15:457-467. 1923. Meiklejohn, J. Aerobic denitrification. Ann. Appl. Biol. , 27:558-573. 1940. Miyake, K. On the nature of ammonification and nitrification. - Soil Sci. 2:481-492.. 1916. Penman, H. L. Gas and vapour movements in soil, I. The diffusion of vapours through porous solids. Jour. Agr. Sci. 30:437-462. 1940. Pulley, H. C., and Greaves, J. D. -An application of the auto- catolytic growth curve to microbial metabolism. Jour. Bact. 24:145-168. 1932. Quastel, J. H., and Scholefeld, P. G. Biochemistry of nitrification in soil. Bacteriol. Rev. 15:1-53. 1951. Raney, W. A. Field measurement of oxygen diffusion through soil. .Soil Sci. Soc. Amer. Proc. 14:61-64. 1950. Rider, B.-F. and Mellon, M. G. Colorimetric determination of nitrites. Ind. Eng. Chem. A. E. 18:96-99. 1946. Russell, M. B. ~Soi1 aeration and plant growth. In B. T. Shaw, Ed., - Soil Physical Conditions and Plant Growth. Academic Press N. Y. pp. 253-301. 1952. Sabey, B. R., Frederick, L. R. , and Bartholomew, W. V. Influence of temperature on nitrification in soils. - Soil Sci. Soc. Amer. Proc. 20:357-360. 1956. Sabey, B.. R., Frederick, L.-R. and Bartholomew, W. V. The formation of nitrate from ammonium nitrogen in soils: III. Influence of temperature and initial population of nitrifying organisms on the maximum rate and delay phase. Soil Sci. Soc.e Amer. Proc. 23:462-465. 1959.. 36p 37.- ESoil Sci. 85:1-9. 1958. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 70 Schloesing and *Muntz, Recherches sur la nitrification. In Compt. Rend. Acad. Sci. (Paris) 89:1074—1076. 1879 .. (Cited by Quastel and Scholefeld, 1951). Sohn, J. B. and Peech, M. Retention and fixation of ammonia by soils. Snedecor, G. W. -Statistical Methods, 4th ed., The Iowa State College Press, Ames, Iowa. Soulides, D. A., and Clark, F. E. Nitrification in grassland soils. -Soil Sci. Soc.~Amer. Proc. 22:308-311. 1958. Stojanovic, B. J. and Broadbent, F. E. Imobilization and minerali- zation rates of nitrogen during decomposition of plant residues in soil. . Soil Sci. Soc. Amer. Proc. 20:213-218. 1956. Taylor, S. A. Oxygen diffusion in porous media as a measure of soil aeration. Soil Sci. Soc. Amer. Proc. 1949. 14:55-61. 1950. Theron, J. J. The influence of plants on the mineralization of nitrogen and the maintenance of organic matter in the soil. Jour. Agr. Sci. 41:289-296. 1951. Thimann, K. V. The Life of Bacteria, 1955. The Macmillan Co. , N. Y. Van Bavel, C. H. M. 'A soil aeration theory based on diffusion. ~Soil Sci. 72:33-46. 1951. Van Doren, D. M. Jr. Relationships between oxygen diffusion rates, as measured with the platinum microelectrode, and plant growth. Ph. D. Thesis, Michigan State University. 1958. Van Doren, D. M. Jr. and Erickson, A. E. Technique for measuring the rate of oxygen diffusion through the soil with the platinum micro- electrode. Manuscript. Waksman, S. A. Principles of Soil Microbiology, 1927. The Williams and Wilkins Co. , Baltimore. 1 Webley, D. M. ~A technique for the study of oxygen availability to micro-organisms in soil and its possible use as an index Of soil aeration. Jour. Agr. Sci. 37:249-256. 1947. APPENDIX 72 Tabulation of pH values obtained from 1:1 soil to water suSpensions from each tube incubated. Table 12. Incubation time (days) Repli- Treat- ment 28 35 40 43 46 49 54 57 63 14 cate 3 3 3 9 5. 5.4 3 5 4 8 7 7 5.6 5. 5 5 5 7 5. 7 7 5 8 . 9 9 5. 5 5. 0 9 1 1 6. 1 1 0 2 1 2 1 2 3 3 5. 5. 5 5 7 5. 5. 5. 5. 5.4 5. 5. 5. 6. 6. 6. 5.8 5. 5. 5. 5. 5.8 5. 6. 92 56 5 7 5. 5.8 5. 5.9 5 5.9 5 5. 58 6.0 5.9 6.1 6.2 6.2 6. 4 4 3 5. 5 5 5 8 . 0 5. . 7 9 9 5 5 4 4 5.6 5. 5. 7 8 7 5. l 0 O 3 3 0 1 2 26. 2 0 3 6.4 5. 5. 5. 5. 5.6 5. 5. 6. 5.. 5. 5 0 1 2 5. 6 5. 5. 6. 6. 6. 5. 5 9 9 0 5. 6. 6. 7 8 9 3 6. 5. 6.4 . 7 5. 5.8 2 2 6. 6. 5. 6. 6.4 6. 5. 6. 3 8 8 6. 6. 5. 6.5 6.3 6.4 6.0 2 6. 5 3 8 7 5. 7 5 5.6 5. 5.6 5. 5. 5. 7 8 5. 6. 0 1 0 6. 2 1 6.4 7.. 5. 5 5. 5 5. 5. 5.4 5.8 5.4 5.8 5.5 5.4 5. 6.2 6. 5.9 6.2 6.0 6. 6.6 6.1 6.0 6.0 6.2 6.0 6. 1 0 2 l 4 6.0 6.0 0 6.0 6. 1 l 6.0 6. 5 7 1 1 6.0 6.0 6. 2 Incubation time (days) 6.0 6. 6.0 6. 28 35 40 43 48 54 57 63 68 6. 14 6.0 6.3 5.8 5.5 5.4 5.3 5.5 5. 3 2 3 6.4 5.4 6.0 5. 5 . 5 5. 6.5 5.6 5. 5. 6.0 6. 5. 5.3 5.3 6.4 5 5 6.3 5.7 6.0 6.1 3 6.6 6.6 6.4 l 6.3 6. 1 6.0 6.3 6.0 6. _6.6 6.5 6.6 6.5 6. 2 6.3 6.1 6.4 6.2 6.5 6.4 5 l 2 6.1 6.1 6.1 6. 6 . 4 Incubation time (days) 36 6. 6.6 6.6 57 64 9O 50 25 18 7. .0 6.9 9 8 . 7 6. 7 I357 0 7.1 7. 6.7 7. 6 5 6.8 0 7.0 7. 7. 6.9 7. 6.8 Continued '9 Table 12 - continued Incubation time (days) 90 64 57 50 36 25 Repli- cate Treat- ment 18 7.0 l 6.8 6.8 6.5 III» 6.8 6.9 6.6 6.6 8 4 6. 0 0 7.1 O l 6.8 6.8 6. 7. 2 6.9 7.1 7 7 7 I‘M 7. 6.8 l 7. 6.8 7.0 6.7 6.9 1 0 0 MM 1 2 7. 7.2 6. 7. 7. 6.8 7. 6.8 Incubation time (days) 34 41 44 47 50 50 53 5. 5. 5. 25 18 53.6 6. 5. 2 1 2 8 5. 5 5 5 5 5 3 4 8 5. 7 1 1 9 3 1 2 3 3 3 2 3 2 9»: 5.5 5. 5 6.0 5. 4 4 8 7 7 5. 0 6.0 1 5.4 6.0 5. 5. 5. 5. 5. 5. 5. 5. 6.0 5. 5. 5.8 7 6. [014' 3 0 0 6.0 5. 6.3 6.4 6. 6. 6. 5.8 5. 6.0 6.3 6. 6.4 6. Incubation time (days) 58 47 50 50 53 5. 44 25 34 18 41 58 6.1 54 55 5.3 53 3 8 8 5.6 4 5.7 6 5. 5 54 5.5 59 .7 5 6.1 6.1 60 6.2 62 60 6.3 60 65 6.4 64 65/0 666 //16 7 7 6.7 6.4 6.8 6.2 2 2 6.4 6.5 6. 6.6 6.8 5 6. 6.6 6.5 6.8 6.6 6. 6.0 6.4 6.4 6.6 6.8 7. 6.6 6.5 6.1 6.7 6.8 6.8 74 Table 13. Tabulation of measured oxygen diffusion rates obtained from each incubated sample with time. Treat- Repli- Incubation time (days) ment Cate 14 28 35 40 43 46 49 54 57 63 gms 02 x 10"8 cm' min" 1 a 32.7 37.1 51.7 52.7 39.5 37. 1 30.7 38.6 54.2 48.8 b 33.7 50.3 55.1 48.8 44.4 28.3 52.2 38.1 37.6 43.9 c 33.2 38.1 43.4 30.7 39.0 36.1 34.6 -- 36.1 49.3 2 a 53.2 53.2 40.0 52.7 47.3 54.2 31.2 43.9 54.2 40.0 b 42.0 33.7 42.5 49.3 44.4 39.0 31.7 44.4 33.7 50.. 3 C 35.6 36.6 34.2 42.0 32.2 33.7 57.6 42.9 45.9 39.5. 3 a 33.7 49.8 40.0 47.8 30.7 38.1 52.7 30.7 34.2 40.0 b 55.6 47.8 17.6 32.7 49.8 30.7 62.5 43.9 46.4 41.0 c 46.8 50.3 29.3 53.7 22.9 36. 1 28.3 22.9 30.1 27.8 4 a 42.9 55.6 40.5 35.1 29.3 49.8 53.7 45.4 28.8 47.8 b 23.9 22.4 28.3 29.3 51.7 26.8 43.9 27.8 56.1 37.6 ‘c 24.9 31.7 32.7 45.4 16.6 37.1 30.7 34.6 38.6 38.6 5 a 23.4 24.4 34.2 15.6 33.2 32.7 13.7 41.5 44.9 49.8 b 37.6 29.3 30.3 27.3 27.8 46.4 28.8 40.0 38.6 34.6 c 44.9 25.4 30.7 35.6 51.7 57.6 40.0 22.9 45.4 31.2 6 a 17.6 25.9 30.7 32.2 39.5 15.1 30.7 26.4 27.8 25.4 b 29.8 37.1 38.6 37.1 38.1 24.4 32.2 33.2 37.1 5.4 c 21.5 6.8 38.1 30.7 40.5 39.5 49.3 35.1 39.0 43.9 Incubation time (days) 14 28 35 40 43 48 54 57 63 68 7 a 12.7 26.4 25.8 11.2 16.6 30.3 21.5 29.3 44.4 38.1 b 18.5 27.3 19.5 24.9 5.9 19.0 31.7 34.2 25.9 39.0 c 26.4 24.4 20.9 21.0 22.0 24.9 30.3 21.5 27.8 23.4 8 a 26.4 39.0 36.1 28.3 30.3 37.6 25.9 23.9 38.6 28.3 b 35.1 19.0 32.7 14.2 29.3 28.3 23.4 23.9 21.5 24.4 c 23.4 12.2 23.4 21.0 22.4 36.6 25.9 24.9 26.4 24.4 Incubation time (days) 18 25 36 -- 43 -- 50 57 64 9g [39' a 26.4 14.6 7.8 -- 5.4 -- 5.9 4 4 5.9 6.3 b 3.9 4.4 19.0 -- 7.8 -- 3.9 5.9 4.9 8.8 c 5.4 -- 7.3 -- 11.2 -- 9.3 5.4 6.3 5.9 Continued Table 13 -'Continued 75 : w Treat- Repli- Incubation time (days) ment cate 18 25 36 -- 43 -- 50 57 64 90 gms O; x 10“8 cm"7‘ min“l MM a 9.3 7.3 13.2 .. 5.4 -- 3.4 7.8 6.3 4.4 b 2.4 1.5 3.9 -- 2.0 -- 1.5 7.3 4.4 2.4 c 14.2 -- 2.9 -- 1.5 -- 1.5 2.0 3.9 13.7 III/1' a 21.0 .4 9.3 _- 4.4 —- 5.4 4.9 4.9 10.2 b 5.9 2.4 23.9 -- 7.8 -- 3.4 3.9 4.4 10.7 c 10.7 -- 6.8 -- 6.8 -- 13.7 16.1 4.9 29.3 [61¢ a 2.4 2.4 5.9 -- 2.9 -- 4.9 4.4 18.1 4.9 b 2.9 2.0 2.9 -- 2.0 -- 1.0 4.4 5.9 18.1 c 2.4 -- 2.9 -- 1.5 -- 1.0 1.0 3.9 25.9 Incubation time (days) 18 25 34 41 44 47 50 50 53 57 9 1’3 a 47.8 30.3 17.1 30.7 -- 24.4 18.1 10.7 28.8 24.4 b 23.9 -- 16.6 20.0 23.9 29.3 23.4 19.5 32.7 22.4 c 22.4 -- 29.3 29.3 28.8 20.5 28.3 18.5 27.8 32.2 [0 IA a 26.8 24.9 36.1 45.4 31.2 26.4 23.4 22.9 39.0 9.8 b 32.7 -- 24.4 32.7 27.3 21.0 32.2 21.0 28.8 33.7 c 30.3 -- 2.4 25.9 35.6 26.4 21.0 23.9 24.9 31.7 Incubation time (days) 18 25 34 41 44 47 50 50 53 58 [/15 a 18.1 29.3 29.5 8.8 8.3 17.6 13.2 13.2 15.1 24.4 b 16.1 -- 6.8 22.9 11.7 5.9 6.3 10.7 12.2 14.2 c 14.6 -- 17.1 13.2 9.8 10.2 12.7 6.3 23.9 18.5 A116 a 13.7 4.9 4.9 27.3 24.4 22.4 9.8 2.0 15.1 5.4 b 9.8 -- 5.4 2.9 20.0 3.4 6.3 7.8 5.4 2.0 c 2.0 -- 2.9 25.9 6.3 2.9 3.9 13.2 2.9 5.4 Table 14. Tabulation of nitrate nitrogen values for each incubated sample. Treat- Repli- Incubation time (days) ment cate 14 28 35 4O 43 46 49 54 57 63 PPM nitrate nitrogen 1 a 7 84 91 73 86 98 106 122 146 158 b 35 77 56 52 48 69 110 24 126 1.51 c 18 35 4 56 57 109 138 -- 138 158 2 a 38 24 53 66 95 102 116 81 84 105 b 39 45 50 65 88 102 93 90 84 87 c 32 38 46 71 96 92 84 98 82 44 3 a 4 25 14 21 65 94 69 97 142 130 b 70 0 56 46 90 102 89 146 138 171 c 66 63 14 18 20 61 24 102 138 174 4 a 13 24 22 71 69 53 55 71 70 62 b 32 17 46 46 42 67 88 56 70 61 c 29 49 43 48 65 48 54 67 60 82 5 a 49 0 32 21 90 41 118 130 118 110 b O 0 70 66 57 98 85 154 138 142 c 0 1 17 66 82 122 49 102 106 126 6 a 13 18 39 48 66 17 69 84 52 75 b 64 13 30 44 58 67 55 74 41 59 c 11 ll 39 54 92 41 64 67 50 71 Incubation time (days) 14 28 35 4O 43 48 54 57 63 68 7 a 7 0 53 4 101 142 122 134 134 69 b 0 25 39 39 49 98 134 114 85 102 c 21 21 77 59 98 114 142 138 199 69 8 a 8 6 61 4O 33 66 27 45 27 27 b 0 33 63 33 37 53 65 23 15 28 c 0 8 36 46 67 66 79 67 51 88 Incubation time (days) -- 25 36 -- 43 -- 50 57 64 90 [33' a -- 2 0 -- 4 -- o o o 4 b -- 21 O -- 7 -- 7 0 0 0 c -- -- ll -- 14 -- 0 0 14 8 Continued Table 14 -‘ Continued 77 Treat- Repli- Incubation time (days) ment cate 18 ‘ 25 36 -- 43 - - 50 57 64 90 PPM nitrate nitrogen N .w a -- 7 9 -- 2 —- 1 O o O b -- 7 7 -— 3 -- 0 O 0 0 c -- -- l -— 6~ -- 0 0 0 6 l5 14 a —— 14 35 —- 14 -- o o o 73 b -- 42 ll -- 14 -- 4 -- 0 4 c -- -- 0 -- 0 -- O O 0 12 ’4 1/2 a -- 4 1 -- 0 -- 2 0 0 3 b -- O 1 -- 4 -- 0 O 0 2 c -- -- 1 —- 4 —- 0 0 0 2 Incubation time (days) 18 25 34 41 44 47 50 50 53 57 9 V3 a 33 16 49 81 -- 116 178 144 161 172 b 47 -- 39 133 147 133 161 158 214 109 c 35 -- 81 67 130 210 165 140 186 203 ID 14 a 98 6O 95 112 94 70 120 53 113 O b 39 -- 82 85 161 11 60 83 100 131 c 28 -- 0 106 74 112 107 114 123 168 Incubation time (days) 18 25 34 41 44 47 50 50 53 58 H 16 a -- 14 53 ~74 70 126 88 133 98 133 b -- -- 45 77 73 144 116 126 165 88 c 12 -- 74 56 O 126 126 133 144 147 [1 16 a 9 o 2 24 54 61 25 2 7 o b -- -- 3 o 66 6 13 7 4 0 c -- -- 4 50 41 30 _ 19 18 1 6 78 Table 15. Tabulation of ammonium nitrogen values for each [incubated sample with time. Treat- 'Repli- Incubation time (days) ment cate 14 28 35 40 43 46 49 54 57 63 PPM ammonium nitrogen 1 a 266 186 291 252 342 215 158 333 142 137 b 227 213 329 308 308 183 308 305 138 46 c 268 224 503 354 264 211 300 --- 134 105 2 a .33 . 29 25 11 3 4 1 1 2 o b 32 20 21 13 2 5 ‘1 3 1 1 1 c 35 25 3o 15 7 3 2 3 2 6 3 a 217 210 256 357 428 251 394 232 110 162 b 256 234 234 238 338 191 284 219 122 142 c 242 242 238 385 345 166 378 228 118 85 4. , a 29 34 3O 6 17 16 6 4 2 6 b 36 33 31 26 28 7 2 7 4 4 c ' 37 18 22 9 24 6 17 7 2 3 5 a 270 347 308 245 196 301 163 138 203 151 b 378 339 248 224 215 187 152 199 171 97 c 304 364 346 207 281 162 235 154 162 122 6 a 29 38 22 8 9 26 ' 2 6 2 2 b 31 4O 16 15 14 8 3 6 4 3 c 32 32 12 10 9 18 3 6 7 11 Incubation time (days) 14 28 35 40 43 48 54 57 63 68 7 a 308 620 168 186 102 61 142 8 20 --.. b 277 354 426 141 346 46 191 151 93 20 C' 420 375 112 228 138 475 134 151 131 93 8 a 39 48 17 15 37 5 21 11 2,3 15 b 38 36 20 26 24 5 2 18 18 9 c 40 38 27 36 6 7 2 1 ll 0 Incubation time (days) ,3 19’ a 297 268 266 -- 269 —- 252 262‘ 217 2, b 282 266 266 -- 245 -- 221 269 214 2:: 2 2 -- ~256 -- 2 .. . i“ Continued Table 15 - Continued T reat- ‘ Repli- t w;— Incubation time (days) 79 ment cate 18 25 36 -- 43 -- 50 57 64 90 PPM ammonium nitrogen l4 W a 75 51 42 .— 38 -- 47 48 46 50 b 37 51 41 -— 38 —- 48 45 43 50 c 33 _— 42 —- 37 -- 55 46 41 60 I! M a 248 268 193 —- 280 —- 287 294 196 53. b 254 256 242 .- 286 —— 256 234 175 207 c 254 .. 256 H 294 —— 294 322 182 228 II. M a 60 65 50 -. 48 -— 44 51 51 58 b 58 68 48 —— 39 -- 51 53 46 50 c 63 __ 54 -— 39 -- 48 52 47 58 Incubation time (days) 18 25 34 41 44 47 50 50 53 57 9 123' a 297 318 222 273 .. 140 165 185 161 168 b 308 -— 228 221 186 193 151 164 119 137 c 293 .. 228 273 213 116 168 116 136 130 [a 1.4 a 46 51 11 6 13 1 1 1 5 53 b 49 .. 14 2 3 29 17 18 3 3 c 40 -- 41 3 4 1 7 1 3 3 Incubation time (days) 18 25 34 41 44 47 50 50 53 58 II 15 a 282 295 245 200 172 130 129 95 101 77 b 315 -- 242 189 133 137 119 122 112 144 c 296 -- 242 189 207 140 112 108 108 88 [1 16 a 53 65 25 15 5 4 4 15 23 6 b 54 —- 42 27 4 8 10 12 18 5 c 56 -- 43 9 8 5 8 10 15 3 8O 18:3 flow 6.? mi. ms; T? 9% WE mam TS. mdo SA odm men 6.; NZNM Wmm m.~m Nam m.om «71$ 93 «6.3 3:5. 6.3.. 26.13 41mm 6.6.“. v.3 6.3 T: 6.2 6.3 92 43$ 7?. man Nam 93 oi man 93 m3 TS 18 WE mac N; oéa 0.3 oém oi 6.3. 53. 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