Short-Lived Radionuclides in Chemistry and Biology - ACS Publications

15 Use of N in Studies of Denitrification 13

J. M . TIEDJE, R. B. FIRESTONE , M . K. FIRESTONE , M. R. BETLACH , H . F. KASPAR , and J. SØRENSEN 1

Downloaded by UNIV ILLINOIS URBANA-CHAMPAIGN on March 5, 2017 | http://pubs.acs.org Publication Date: March 1, 1982 | doi: 10.1021/ba-1981-0197.ch015

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Departments of Crop and Soil Sciences, Microbiology and Public Health, and Cyclotron Laboratory, Michigan State University, East Lansing, MI 48824

Denitrification results in major losses of fertilizer and native nitrogen from soil, fresh water, and marine habitats. The radioactive isotope, N, allows direct measurement of N , the principal denitrification product, in any atmosphere and provides a very sensitive measure of denitrification activities. The findings of laboratories that have used N in denitrifi cation studies are summarized in this chapter. Emphasis is placed on the methods for production, purification, and detection of N forms important to denitrification investiga tions. The summary of research results includes information on the following: measurement of denitrification rates, iso tope dilution experiments to investigate denitrification inter mediates, factors influencing the proportion of N O vs. N produced, and the partitioning of nitrate between denitrifi cation and dissimilatory nitrate reduction to ammonium for bacterial cultures and natural samples. N and N are considered complementary methods, since each is uniquely suited to certain aspects in the study of denitrification. 13

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TTVenitrification is the process by which nitrogenous oxides, principally -"-^ nitrate, are reduced to gaseous nitrogen products, primarily dinitro gen and nitrous oxide. The process is carried out by a diverse group of bacteria that have the unique capability of using nitrogenous oxide as Current address: * Current address: * Current address: * Current address: ' Current address: 1

Lawrence Berkeley Laboratory, Berkeley, CA 94720. University of California, Berkeley, CA 94720. NASA Ames Research Center, Moffet Field, CA 94035. Cawthron Institute, Nelson, New Zealand. University of Aarhus, Aarhus, Denmark. 0065-2393/81/0197-0295$05.00/0 © 1981 American Chemical Society

Root and Krohn; Short-Lived Radionuclides in Chemistry and Biology Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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alternative electron acceptors for their respiratory metabolism when oxygen is limiting. Bacteria capable of this process are widespread. Thus it is not surprising that denitrification is well known in soils and sediments of all types but can also occur in more localized habitats such as food products, animal intestinal tracts, and waste treatment facilities. The pathway of denitrification is generally thought to be N 0 ~ - » N 0 " - » NO - » N 0 - » N (I) with N O the only intermediate in dispute (2,3,4). Since denitrification produces N from a nitrogen form available to plants, it represents a loss of this scarce resource for biological growth and productivity. In effect, denitrification is one-half of the nitrogen cycle, the other half being nitrogen fixation, the process that converts N to nitrogen forms available to organisms. It is estimated that about one-fourth of nitrogen fertilizer is lost by denitrification (5); and on a worldwide basis, estimates of total global denitrification are 200 million metric tons (6,7). With the projected shortages of food and energy, this loss is one that all would like to reduce. Besides the major concern over the loss of productivity, there are four additional reasons why the denitri fication process is of concern: (i) nitrous oxide has been implicated in destruction of the ozone layer (8,9) and in warming of the planet through the greenhouse effect (10); (ii) nitrite, an intermediate in the process reacts with secondary and tertiary amines to form nitrosamines, some of which are among the most potent carcinogens known (11); (iii) nitric oxide, which also seems to be produced during denitrification, is a free radical and can react with heme compounds and other cellular constituents, and thus N O may have some detrimental impact on orga nisms; and (iv) the denitrification process can be used to remove nitrogen from waste that otherwise would produce an excessive nutrient load in a receiving habitat (12). Despite the obvious importance of denitrification to human beings and to the environment, it remains the least understood process in the nitrogen cycle. One major reason for this is the absence of methodology to measure the process directly and sensitively, because the product, N , cannot be measured in the presence of its atmospheric background. The radioisotope, N , therefore provides a major advantage, because it can be sensitively detected in the presence of any atmosphere. A second advantage of N is that its high sensitivity allows one to measure rates of denitrification by addition of the tracer without affecting the natural nitrate concentration. The high sensitivity of N is apparent from Table I, which has a comparison of the N detection limits to those of other isotopes commonly used by biologists. Generally, one need not add more than femtogram quantities of N 0 ~ (e.g., 68 fg = 0.1 mCi, which is usually sufficient). Other advantages of N are that kinetics at very low N substrate concentrations can be investigated, that the gamma emission 3


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Table I.

Comparison of Sensitivity of

Isotope 13 15 C N

N

1 4


N in Denitrification Studies

ssg H 8

297

13

1 3

N with Other Isotopes

Half-life

Maximum Specific Activity Possible (mCi/m atom)

10 min stable 5730 yr 14.3 day 87.4 day 12.35 yr

1.4 X 10 (99 atom % excess) 62.4 9.1 X 10 1.5 X 10 2.9 X 10* 10

6

6

Minimum Detectable Amount (Moles)* 3 7 7 5 3 1

X 10" X 10" X 10' X 10"" X 10x 10" 20

9>

12

16

• Assuming 1000 dpm minimum detectable radiation. * Assuming 0.001 atom % excess of N and 1 mg N gas produced in 10 mg N gas sample to be the minimum detectable N enrichment. However, the sensitivity can be increased approximately two orders of magnitude by using the new quadrupole GC mass spectrometers, due to their greater absolute sensitivity (#). 1 5

1 5

allows nondestructive measurement of biological materials (of special value in studies which include plants), and that one can locate and measure small pools of intermediate N species, as in the isotope dilution experiments discussed later. We are aware of four groups that have conducted denitrification studies using N : our group at Michigan State University, which uses the sector-focused cyclotron on campus; Gersberg and Goldman cooper ating with Krohn and others at the Crocker Nuclear Laboratory at the University of California, Davis; Stout of the New Zealand Soils Bureau cooperating with McNaughton and More using the Van de Graaff accelerator at the DSIR Institute of Nuclear Science near Wellington, New Zealand; and Hollocher of Brandeis University cooperating with Cooper and Tilbury using the cyclotron at the Memorial Sloan-Kettering Cancer Center in New York City. There are many more facilities capable of producing N than are being used for research on the nitrogen cycle. The major discouragement to expansion of N use will probably remain the rather lengthy development phase and the need for collaboration among several people of very different skills. 1 8

1 8

1 3

Experimental The Gas Stripping System. This system has been the most popular for denitrification studies probably because it provides the most immediate and sensitive measurement of denitrification rates. It was first used by Gersberg et al. (13,14) to measure denitrification rates in rice paddy soils, sediments, and hypolimnion water, and later by Tiedje et al. (15) and Stout and More (16) to measure denitrification rates in soils. A schematic of the system used by our group is shown in Figure 1.



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A l l three groups used helium to sweep the N gases produced to the traps. Gersberg et al. (13) used a single trap of Molecular Sieve 13 X immersed in liquid nitrogen to collect all N gases. Tiedje et al. (15) and Stout and More (16) used differential trapping procedures to separate N 0 and N so that the rate of production of each gas could be measured. We used liquid nitrogen to freeze out the N 0 but not the N (Figure 1), while Stout and More used a warm molecular sieve trap to retard the N 0 but not the N . Stout and More had to correct N 0 counts for the transience of N . We found that the N retention in our N 0 trap was less than 3% and thus was considered insig nificant. AU groups have used N a l ( T l ) detectors placed adjacent to the traps and similar electronics to monitor N accumulation in the traps. We also have on-line capability for correction of half-life, background and detector calibra tion to a standard source ( N a ) (15). The general limitation of the stripping system is that it requires helium as the sweep gas since larger-molecular-weight gases seem to cause channeling in the cold sieve trap, thereby breaking its trapping integrity. Since helium must pass through the sample, the gaseous environment of the sample changes. For naturally anaerobic samples, this is not a concern, but many denitrifying habitats have some oxygen present, for example, most soils and the aerobicanaerobic interface of sediments. In the latter cases, the oxygen status can not be maintained with a helium sweep gas, and thus one would expect that denitrification would be affected since oxygen is a major environmental regu lator of this activity ( I ) . Stout and More (6) have added N 0 " to natural soil cores and used helium as the sweep gas to pass over the core surface, but they have not completed enough studies nor have they characterized the effect of helium on the soil's gaseous environment to be able to judge the appropriate ness of this procedure for measurement of natural denitrification rates. A stripping system that allowed use of some oxygen and yet trapped all the N would be much more useful for studies aimed at determining natural denitrification rates. 1 3

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N Gas Analysis. Our laboratory has developed a gas chromatography (GC) system that allows immediate measurement of the radioactivity in each N gas peak by coupling a proportional counter (PC) to the effluent of the microthermister detector cell (15). The output is fed to a computer and the data are later corrected for half-life, background, and the area under the peaks is integrated. Further details on the electronics and the data analysis program are presented in detail elsewhere (17,18). Measurement of NO is problematic due to its reactivity. Extra N O is added to syringes before taking samples in which N O is to be measured, providing extra carrier to reduce N O loss by adsorption. One gas separation system used was Poropak Q and Molecular Sieve 5A columns in series in which the N and NO were separated on the molecular sieve (15). This system was used successfully, but the NO tailed badly and showed some memory on the column. A second system used a liquid nitrogen cooled loop between the injector and the Poropak Q column (19). N 0 and NO are retained in the cold loop while N is measured; the loop is then warmed and N 0 and NO are separated and measured. This seems to be a more accurate system, but it requires more manipulations. An alternative that we used in the NO label exchange experiment was to avoid the difficulty of the NO and N separation by choosing organisms that lacked the capacity to reduce N 0 to N (4). In this case, only N 0 and NO gases were present and could be analyzed rapidly on the single Poropak column. A GC system is essential to verify the identity of the gases and can be used to analyze N in any atmosphere. We have also used it to obtain denitrifica tion rates analogous to those from the stripping system. However, the GC method is more time-consuming, yields fewer points, and is much less sensitive since only a small subsample of gas is taken. N Ion Analysis. A radio high performance liquid chromatograph (R-HPLC) was developed in our laboratory to monitor N H , N 0 " , and N 0 " in studies of dissimilatory nitrate reduction (15). The system was patterned after that of Tilbury and Dahl (20). Separation was accomplished in 5 min on a strong anion-exchange column (Partisil SAX) with a pH 3.0, 0.05M phos phate buffer. Detection was by y coincidence counting using 2 Nal(Tl) crys tals. Data were collected, corrected, and reported as for the gas chromatography-proportional counting system above. An example of a separation is shown in Figure 2 for a N source and for the products following microbial reduction of that substrate. Our group used the R-HPLC to monitor the N species in each source (since this varied somewhat), to prove purity after a purification procedure, to measure temporal changes in the ions after incubation with bacterial cells or natural samples, and to quantify the ions inside cells. Cells and debris from natural samples were removed by filtration through a 0.22-/mi filter prior to injection on the R-HPLC column. The internal N species were recovered by extracting washed cells on a filter with hot methanol and by analyzing the methanol extract. The R-HPLC system was more useful in studies in which the dissimilatory fate of nitrate was ammonium rather than dinitrogen and thus sediments, sludge, and rumen contents were those most commonly analyzed. Although this R-HPLC design was very reliable and useful in our studies, we were sometimes limited by the lower sensitivity of the coincidence cir cuitry. It may be advantageous to design future systems with a /? detector to gain sensitivity. Measurement of N Uptake by Cells Assimilating Nitrate. To measure this fate of nitrate, we collected cells incubated with N by passing a portion 1 3

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Figure 2. Separation of NHf, NOf, and N0 - by R-HPLC. The components of an untreated N source are compared to the products of the same source after incubation 13

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with a culture of Pseudomonas (PS

type 19).

TIME (MIN)

of the incubation mixture through 0.45-^m cellulose-acetate membrane filters. The collected cells were then washed with buffer and the filters placed i n scintillation vials with 10 m L of scintillation cocktail (e.g., FilterSolv, Beckman Instruments). The vials were then shaken 10 min to digest the filters and counted twice in a liquid scintillation counter. We devised a system to correct for the N 0 " and F ( * = 110 min) bound to the filter, since washing of filters did not remove these counts. To determine the contribution of F to total radioactivity and to correct for N decay, we solved the following pair of simultaneous equations: 1 3

1 8

3

1 / 2

1 8

1 3

C P M (fa) =2Vo •

+

• D (ti)

(1)

• D (t»)

(2)

F

C P M ( f e ) — No • D*{t») + F

0

r

In these equations, C P M ( * i ) represents the total counts i n the sample vial recorded at elapsed time, t N and F refer to the cpm attributable to N and F , respectively, in the sample at the start of counting the sample set. Dx(ti) refers to the fraction of N radioactivity remaining at time t It is determined by evaluating the expression exp (— In 2 • t/t ), where t is the half-life of N , 9.96 min. A similar expression is represented by D ( * i ) , ex cept that the half-life of F is 110 min. Since the counts per minute and the elapsed time (hence fraction remaining) are known for each sample, the equations need only be solved for N and F . This was done using a program (18) written for an IMSAI 8080 microcomputer in our laboratory. This com puter was connected to the scintillation counter (via a Beckman RS232C Communications Interface) to receive counting data directly. Such a con figuration permitted analysis of an experiment within a few minutes after the last sample had been counted, so that subsequent experiments could be modi fied as needed. Since F has a much longer half-life than N , its contribution to total counts increased continuously after bombardment. In all experiments in which cells had incorporated radioactivity, the contribution of fluorine was lower than v

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it was in the source used, indicating that there was a selective enrichment for N in the filtered cells. In contrast, filters through which samples of the source were passed to measure nonspecific adsorption always had a higher percentage of counts attributable tofluorinethan the source or the filtered cells, indicating F was preferentially bound to the filters. Results of a typical experiment that showed these effects are presented in Table II. An alternative method for measuring N 0 " uptake by bacterial cells is the silicone oil sedimentation method reported on by Thayer and Huffaker in Chapter 17 of this volume. Measurement of Total N Counts. We usually determined total N in samples by placing the sample in Nal(Tl) well counters with data fed to the same computing system as for the above analyses for background and half-life correction (15). Calibration is by a standard N a source dissolved in water and placed in a test tube. The well counters had the most reproducible count ing geometry of any of our detection systems and thus were used to obtain total amount of N source added to any experiment. F present in the samples was corrected by using the two-component decay curve analysis. Calibration of Detectors, Especially GC-PC Against R-HPLC. For many experiments, it was useful to calculate a N balance by adding up each fate of the original label. Since the counting systems we used have different geome tries and detection methods, an isotope balance was difficult to achieve. We cross-calibrated our detection systems in the following way: The gas stripping system, R-HPLC, and well counters were referenced to ^Na, the scintillation counter was referenced to the well counter by using the same N source in each, and the GC-PC was referenced to the R-HPLC by conversion of nitrite to dinitrogen according to the following equation: 1 3

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( N0 ") + N(V + NH S0 H -> N—N + HSCV + H 0 + ( N(V) 13

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A standard N source ( < l m L ) containing N 0 " and N 0 " previously analyzed by R-HPLC was reacted with 0.1 mL of 5% sulfamic acid in a sealed 1 3

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Table IL Corrected Data for Uptake of lfM N0 ~ by Bacterial Cells Grown on NCV and W0 ", Illustrating F Contribution to Total Radioactivity on Filters 13

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Incubation Time (Min)

Disintegrations per Minute ' 0

13

N

3

2

18

Percent asp

Percent of Possible N Incorporation 1S

0.5 1.0 2.0 3.0 4.0

1092 1432 1407 2166 1024

18,667 22,756 35,392 41,253 45,643

5.53 5.92 3.82 4.99 2.19

4.56 5.56 8.65 10.08 11.15

Adsorption to filter

347

494

41.27

0.12

144,635

409,303

26.11

100.00

N solution

13

"Values reported as disintegrations per minute since they have been calculated back to the time we began counting the sample set.


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vial. The vial was shaken for a few minutes and then the gas and liquid products were analyzed by GC-PC and R-HPLC, respectively. The original radioactivity in the N0 ~ should equal the total radioactivity in the N — N produced. The N0 ~ is conserved in the reaction and was used to check on the accuracy of the dilution and transfer. The R-HPLC analysis after the reac tion also confirmed that total decomposition of the N 0 " had occurred. After accounting for the appropriate dilutions, we obtained an efficiency ratio be tween the GC-PC and the R-HPLC, thus allowing the nitrogen gases and ions analyzed in an experiment to be evaluated on equivalent terms. This procedure should be done each day the instruments are used, since the GC-PC particularly can easily vary in sensitivity due to slight changes in gas flow and electronic settings for the proportional counter. 13

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Production of

N

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Production of N 0 ~ by the 0 ( p , a ) N Reaction with an H 0 Target. Experimenters with access to cyclotrons appear to have settled on this reaction as the most convenient for production of N 0 ~ , since the target is easy to prepare and labeled nitrate is produced directly and in solution ready for use (13,15). Our yields of N 0 ~ are 10-20 mCi using a 15-MeV proton beam of 1-3 fiA and bombarding for 10-15 min. Others obtained greater than 99% of the N as nitrate (13), whereas we typically found the following distribution of N species: 75-95% N0 ~, 5-10% N0 ~, 0.5-25% N H plus traces of N 0 and N (15). These differences in products are probably due to differences in current and target environment, though we were not able to increase reproducibly the proportion of N found as nitrate. We could easily remove the contaminating N H , N 0 , and N by flash evaporation (approximately 3 min) under alkaline conditions (15). Nitrite could be converted to nitrate by H 0 oxidation (15), but this procedure was cumbersome and was not always reliable. This method of N 0 ~ production does yield some F from the O ( p , n ) F reaction due to the natural abundance of 0.2% O in H 0 (15). O depleted water has been used (21) to reduce the F inter ference that can be severe after long counting periods. Since no F gases are formed, this contaminant was not a serious problem in denitri fication studies. N 0 ~ can also be obtained using the C ( p , n ) N reaction with a graphite target developed to produce N — N (22). The advantage of this reaction is that the yield of N is about 10-fold greater. However, the lengthy and complex process of conversion of the N to nitrate makes this reaction less attractive. We have also demonstrated the feasibility of producing N with heavy ion beams and have reported this separately in this volume (Chapter 15). Production of N 0 ~ by C ( d , n ) N Reaction on a L i C 0 Target. McNaughton and More (23) have produced up to 57 fiCi N 0 ~ directly 13

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by this reaction using a 2-MeV, 1-1.3-/JA deuteron beam from a Van de Graaff accelerator and bombarding a L i C 0 pellet for 2 0 min. They recovered the N 0 ~ by mixing the ground pellet with 2.5 mL water and Amberlite 252 ion-exchanger resin in the hydrogen form. The L i was removed by the resin and the displaced H formed hydrogen carbonate. The C 0 was then driven off by heating. They found traces of F , C N and NO«f in their product although at least 8 5 % of the original N was in the nitrate form. This N 0 " source was used by Stout (16) in his studies of soil denitrification. Although the N 0 " yields are 100-fold less with this system than with the 0 ( p , « ) N reaction, these workers have been able to perform denitrification experiments with the gas stripping system. Their success with low-energy accelerators to produce N should be encouraging to others interested in using this isotope, since these machines are more common. Production of Other N Species. Each of the intermediates and products in dissimilatory nitrate reduction can be experimentally useful if labeled with N . Procedures for making each of these species are summarized in Table III. We have demonstrated each. The C d reduction procedure to produce nitrite worked well when we used the C d prepara tion procedure of McElfresh et al. (24). However, we used a small 3

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Table III. Methods for Production of N Inorganic Species Useful in Studies of Dissimilatory Nitrate Reduction 1 3

Method

Total Elapsed Time (min)

N0 "

Direct production by bombardment {IS, 15,28)

5

N0 "

Reduction of N 0 " to N 0 " on a copperized cadmium column (19,24) Produced by biological reduction of N 0 " in the presence of N O (see Table IV and Figure 7) Produced by biological reduction of N 0 " by denitrifier species incapable of further N 0 reduction (4)

Desired N Species

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N0

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N—N 0 2

N—N

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N—N

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NH

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Produced by biological reduction of N 0 " using any active denitrifier species Produced by sulfamic acid reduction of N 0 " (see section on detector calibration) Reduction of N 0 " with DeVarda's alloy and distillation to recover the N H

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pencil-sized column and gravity flow. N 0 " was usually the only species detected by R-HPLC if the source was in the column about 3 min and the effluent was treated by alkaline evaporation. Biological methods are suggested for NO, N 0 , and N production, since they are the easiest for someone investigating denitrification as the organisms and procedures are standard. In addition, the biological process provides a high-purity, nontoxic product. Chemical methods usually require a much longer development and would be worthwhile only if extensive use of the product is expected. Both N O and N — N 0 are readily produced, as indicated in Figure 7. In this experiment, a low cell density was used to examine the kinetics of the reaction, but the density could easily be increased 10-fold or more to get a virtually immediate conversion to product. The N O method may be less useful because it yields a low specific activity product due to the sizeable N O addition. 1 3

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Measurement of Rates of

Denitrification

The gas stripping system has been the principal method for assay of denitrification rates by N , since one need only measure the rate of N gas accumulation from a sample in which the native nitrate pool has been amended with tracer quantities of N 0 ~ . If the labeled nitrate is truly carrier-free, then the calculated labeled nitrate added is 10 - to 10 -fold less than the indigenous nitrate pool and thus will not alter the natural denitrification rate. An example of data obtained by the stripping system is shown in Figure 3. These rates were calculated for the linear portion of these curves by multiplying the Adpm N gas • time" • g soil" by the specific activity of the nitrate (fxg N 0 " — N present per total N 0 " added), which is analogous to the equation shown below. Gersberg et al. (13) first demonstrated the usefulness of this system by measuring denitrification rates using a rice-paddy sediment slurry. They obtained a linear pattern of N gas accumulation for the first 10 min after label addition. They calculated the natural denitrification rate using the following equation: 1 3

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5

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7

1

1

1 3

3

3

where D is the rate at which gaseous nitrogen is evolved (fig min" ); dN/dt is the rate of N gas accumulation (fiCi • min" ); N is the decay correction, which is the /*Ci multiplied by A (A. = 0.0693 min" ); N 0 " is the activity of this species in the sample (ftCi); and N 0 ~ is the amount of unlabeled nitrate (fig) in the sample. For rice soils, they obtained reproducible rates of 1-6 ftg-N • L " • h" . 1

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Soil Science Society of America Journal

Figure 3. N gases produced from soil incubated in gas stripping sys tem after adding a mixture of 2.0 ppm NO ~—N (final concentration) and 3.7 mCi NOf/ N0 ' (5 X 10~ ppm) to a soil slurry (15). Control of autoctaved soil shows no labeled gas was produced. 13

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Gersberg (14) also used this system to help resolve why high concentrations of nitrate persisted in the oxygen-depleted bottom waters of Castle Lake. The denitrification rates he measured for the anaerobic water were only 0.003-0.05 /*g-N • L " • h" compared with the sediment rates of 0.3-0.6 ^g-N • L " • h'K We used the stripping system (Figure 1) to obtain denitrification rates in soil slurries and in lake sediments. Slurries were used to obtain rapid mixing of N 0 ~ with the sample and to facilitate efficient stripping of the product gases. This condition probably enhanced the rate of denitrification, especially for soils in which diffusion of nitrate and carbon to denitrifying organisms may have been rate-hmiting due to the low moisture tension and the exclusion of oxygen by the helium stripping gas. Hence, we did not use the system to measure natural rates but to deter mine how various factors altered the rates and ratios of gas products produced. Denitrification rates that we observed ranged from 0.1-2 fig-N • g" • h" for different soils (25,26). These rates are higher than those Gersberg (14) found for the aquatic environment although the values cannot be directly compared since the aquatic ones are on a volume basis (per liter) and the soil data are on a weight basis (per gram). Furthermore, the higher nitrate pool in soils should yield higher denitrification rates. One striking difference between soils and sediments is that the sediments show immediate N gas production, often with total label conversion to product within 10 min; in soils, a 10-12 min lag is repeatedly observed before a linear rate of gas production is noted (15). We did notice that this lag was reduced when N 0 ~ was used, possibly indicating a lag in the nitrate reductase activity in soils. 1

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Stout and More (16) used natural soil cores amended with N ( V but no carrier in 2.5 mL water (see pp. 303) and a helium sweep gas to collect N gases emitted from the soil core surface. Cores were freshly collected, adjusted to desired moisture tension, and amended with denitrifying cultures or sterilized by ^Co irradiation or autoclaving. Autoclaved soil produced some N — N 0 but no N — N , whereas irradiated soil produced both gases. Addition of a denitrifying pseudomonad markedly increased the N — N produced by soil sterilized by both methods but generally reduced the amount of N — N 0 produced by the sterilized soil. The mechanism responsible for the N — N 0 produced in the sterilized soils is not known. We did not observe signifi cant production of this gas in our sterilized soil slurries (Figure 3). In the published report of Stout and More (16), positive rates of N — N gas production were not discernible from natural soils (without bacterial amendment) over the 30-min assay period. However, in later work Stout and Coleman were able to measure N — N and N — N 0 gas production from initially aerobic soil cores and were able to recover up to 50% of the N 0 — N as N gas in some cases (J. D . Stout and D. C. Coleman, poster at International Microbial Ecology Symposium, Warwick, England, 1980). One of the advantages of the stripping system is that it is easy to measure rapid changes in rates of N 0 and N production in response to a change in environmental conditions. Figure 4 illustrates the effect of lowering the p H of a soil slurry from p H 6.7 to 5.2. The rate of N production decreased but total denitrification did not. Due to the immediate response, the effect is thought to be on the enzymes already functioning in this process rather than being an adaptive response to the p H change. Other studies on this same soil (26), show that this change 1 3

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2

2

Figure 4. Pattern of N—N 0 and N—N production from a Brookston soil amended with N0f plus 2 ppm NO ~—N carrier at its natu ral pH (67) and after lowering the pH with HCl to 5.2 at 23 min. Data obtained in the gas stripping system. 13

13

2

2

13

s

10

20 30 TIME (Minutes)

40


50

o


15.

N in DenitHfieation Studies

TIEDJE E T A L .

307

13

Time

[min.]

Figure 5. Pattern of N—N O and N—N production from anaerobic digestor sludge amended with NO " plus 10'* M NCV carrier and mea sured by GC-PC. 13

13

B

2

13

S

in N 0 production was not due to time of incubation. This p H response was observed in the presence of modern nitrate concentrations (in this case, 2 ppm N0 ~—N) but not in the presence of low concentrations of nitrate (27). The above studies have all used the stripping system for measure ment of denitrification rates. We have also obtained similar data using the GC-PC by repeated gas sampling of the incubation vessel. An example of results obtained by this method is shown in Figure 5. Here, 7 g of anaerobic digestor sludge was incubated in a 26-mL serum bottle with N 0 " and 10" M NO

Short-Lived Radionuclides in Chemistry and Biology - ACS Publications

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