OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. ( FACTORS INFLUENCING THE PRODUCTION OF NITROUS OXIDE DURING DENITRIFICATION By Mary Kathryn Firestone AN ABSTRACT OF A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Cr0p and Soil Sciences 1978 ABSTRACT FACTORS INFLUENCING THE PRODUCTION OF NITROUS OXIDE DURING DENITRIFICATION By Mary Kathryn Firestone In the last five years there has been a renewed interest in soil denitrification; this interest has been stimulated by the suggestion that soil evolved nitrous oxide (N20) contributes to the depletion of the Earth's ozone layer. Although it has generally been found that dinitrogen (N2) is the major product of denitrification in soils, under some conditions large quantities of N20 are also produced. The micro- biological factors which promote the production of N 0 rather than N 2 2 are not well understood. The environmental parameters controlling N20 evolution can Operate at the biological level by influencing the produc- tion of N20 versus N2 or at a soil structural level by controlling the diffusion of N20 through the soil matrix. The factors which influence the biological production of N 0 were 2 investigated using l3N labeled N03- and N02- generated by proton bombard- ment of water using the 160(p,a)13N reaction. Use of the radioactive isotOpe allowed very sensitive, direct quantitation of N N O and NO 2’ 2 and permitted experimentation with extremely small additions of electron acceptor (about 68 fg NO --N). 3 Mary Kathryn Firestone 13NO3_ was added to two soils and three denitrifying bacterial cultures so that N 0 from denitrification was 13N-labeled. In bacterial 2 l cultures and in the heterogeneous bacterial flora of soils, the [ 3N]- N20 intermediate exchanged readily and quantitatively with an added pool of nonlabeled N20. This indicates that N20 is an obligatory inter— mediate of denitrification, and exists as a free intermediate capable of diffusion away from the sites of active reduction. This provides some understanding of why environmental factors can influence N 0 production 2 relative to N2 since any factor which produces a change in the relative rate of N 0 reduction compared to the rate of N 2 0 production can result 2 in N20 free to diffuse away from the active site. The presence of other respiratory electron acceptors was found to influence the prOportion of N20 produced during denitrification. Increasing concentrations of NO3 were found to cause prOportional increases in production of N20 relative to N2; but even at the highest concentration of N03- tested (20 ppm NO3--N) N2 remained the dominant product of denitrification. Nitrite had a much greater influence than did N03-. In the two soils examined, the presence of relatively low concentrations of NO - (:_2 ppm NO2 -N) caused N20 to become the dominant 2 product of denitrification. The fact that N02- had a much greater effect than N03- on N20 production suggests that N02“, not NO3-, may be 2 (0-20 ppm N02--N) the overall rate of denitrification did not increase the principle effector. In the range of NO concentrations examined 2 caused a decrease in the rate of N20 reduction to N2. The presence of 02 caused an increase in the N20/N2 ratio as well as a large reduction with increasing N02- availability, indicating that the presence of N0 in the rate of 13N gas production. Mary Kathryn Firestone It was found that temporal changes in the composition of the gaseous products of denitrification occurred following the onset of anaerobic conditions. Dinitrogen (N2) was the major product during the early period of anaerobiosis (O to 3 hours), after which the relative production of N20 increased, resulting in N O as a significant or 2 dominant product until 12 to 32 hours (depending on soil). The net rate of N20 production then declined; this decrease in the prOportion of N20 did not occur in the presence of an inhibitor of protein synthesis, or in the absence of N20 or NO -, indicating that dg_gggg_synthesis of N20 reducing enzymes was induced. When an exogenous carbon source (glucose) was added, the prOportion of N O remained high. 2 The role of nitric oxide (NO) as an intermediate in denitrification was also investigated using 13N03- and 13N02- as substrates. Labeled NO was trapped in pools of nonlabeled NO in both soils and bacterial culture. In soils the production of 13NO from 13N03- was biologically mediated, but sterilized cells produced significant quantities of 13N0 from 13N02... The presence of nonbiological catalysis made it impossible to conclude that NO was an obligatory intermediate in denitrification. To Richard ii ACKNOWLEDGEMENTS The enthusiasm, patience, and knowledge of Dr. James Tiedje have provided the framework for my education as a scientist. I thank him for all that he has contributed, for his friendship, and particularly for encouraging me to pursue my Ph.D. Over the years I have had the good fortune and pleasure of working with a number of outstanding fellow students. Their knowledge, experience, and friendship have allowed me to grow professionally and personally. I thank all of these people but especially Scott Smith and Mike Betlach with whom I have worked most closely. The members of my guidance committee, Drs. B. G. Ellis, A. R. Wolcott, A. E. Erickson, and C. P. Wolk, have each been a teacher, friend, and research advisor to me. In addition, Dr. Wolk should be recognized for his contributions to the develOpment of our 13N research project. I thank Lillian McLean for her super typing and patient good humor through numerous revisions. Finally, without the personal and scientific support of my husband, Rick, literally none of this would have been possible. My graduate study has been supported by an NSF Traineeship and my research by USDA Regional Research Project NE-39 and a grant from the ‘National Science Foundation. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . viii INTRODUCTION AND EXPERIMENTAL OBJECTIVES . . . . . . . . . . . . 1 LITERATURE CITED . . . . . . . . . . . . . . . . 9 CHAPTER I. STUDIES ON NITROUS OXIDE AS A FREE, DIFFUSABLE INTERMEDIATE OF DENITRIFICATION IN SOILS AND BACTERIAL CULTURES C O O C O O O O O O O C O O O 1 3 MATERIALS AND METHODS . . . . . . . . . . . . . . 14 RESULTS . . . . . . . . . . . . . . . . . . . . 18 DISCUSSION . . . . . . . . . . . . . . . . . . . 26 LITERATURE CITED . . . . . . . . . . . . . . . . 33 CHAPTER II. THE INFLUENCE OF NITRATE, NITRITE AND OXYGEN CONCENTRATIONS ON THE COMPOSITION OF THE GASEOUS PRODUCTS OF DENITRIFICATION IN SOIL . . . . . . . 35 MATERIALS AND METHODS . . . . . . . . . . . . . . 36 RESULTS . . . . . . . . . . . . . . . . . . . . 40 DISCUSSION . . . . . . . . . . . . . . . . . . . 58 LITERATURE CITED . . . . . . . . . . . . . . . . 62 CHAPTER III. TEMPORAL CHANGES IN COMPOSITION OF GASEOUS PRODUCTS OF DENITRIFICATION FOLLOWING ONSET OF ANAEROBIC CONDITIONS 0 O O C O C C O O C O O O C 63 MATERIALS AND METHODS . . . . . . . . . . . . . . 64 RESULTS . . . . . . . . . . . . . . . . . . . . 67 DISCUSSION . . . . . . . . . . . . . . . . . . . 82 LITERATURE CITED . . . . . . . . . . . . . . . . 87 CHAPTER IV. INVESTIGATIONS ON NITRIC OXIDE AS AN INTERMEDIATE IN DENITRIFICATION I O O I I I O O O O O O O I O 89 MATERIALS AND METHODS . . . . . . . . . . . . . . 91 RESULTS . . . . . . . . . . . . . . . . . . . . 93 DISCUSSION . . . . . . -.. . . . . . . . . . . . 101 LITERATURE CITED . . . . . . . . . . . . . . . . 102 ii: APPENDIX METHODS FOR THE PRODUCTION AND USE OF STUDIES OF DENITRIFICATION . . . . . ISOTOPE PRODUCTION AND PURIFICATION . ANALYTICAL PROCEDURES . . . . . . . DATA ACQUISITION AND ANALYSIS . . . . LITERATURE CITED . . . . . . . . . . Page 106 106 110 123 126 Table 10 11 12 13 LIST OF TABLES Exchange of [13N]-N 0 produced by Pseudomonas fluorescens and Flavobacterium sp. with a nonlab81€d N 0 p001 O o o o o o o o o o o o o o o o 2 13N-gas production by Pseudomonas 151, Alcaligenes sp. and Micrococcus sp. . . . . . . . . . . . . . 13 N ionic species found after incubation of 13N substrate with Pseudomonas 151, Alcaligenes sp. and Micrococcus sp. . . . . . . . . . . . . . . . Exchange of [13N]-N 0 produced by a Brookston soil with a nonlabeled N20 pool . . . . . . . . . . . . Exchange of [13N]—N 0 produced by a Spinks soil With a nonlabeled p001 O O O O O O O O C I O O O 0 Characterization of soils . . . . . . . . . . . . Effect of N02- concentration on NZO/N2 . . . . . . Effect of N03- concentration on NZO/N2 . . . . . . Effect of 0 on denitrification activity and products in a Brookston soil . . . . . . . . . . . Influence of 0 concentration on denitrification activity and products in a Miami soil . . . . . . Rates of N 0 production and denitrification following ghe onset of anaerobic conditions in a Brookston soil . . . . . . . . . . . . . . . . . Rates of N 0 production and denitrification following Ehe onset of anaerobic conditions in a Miami 8011 O O O O O I O O O O O I O O O O O O I 0 Rates of N 0 production and denitrification in the presence and absence of chloramphenicol in a conover Soil 0 O I O O O O O O O O O O 0 O O O O 0 vi Page 19 22 23 28 29 37 46 48 53 57 71 73 74 Table 14 15 16 17 18 19 20 21 22 23 Page Rates of N 0 production and denitrification in the presence and absence of chloramphenicol in a Brookston soil . . . . . . . . . . . . . . . . . . 76 Rates of N 0 production and denitrification_after anaerobic ncubation with N0 , without N03 , and with N20, in a Brookston soiI . . . . . . . . . . . 77 The effect of glucose addition on the rates of N 0 production and denitrification in a Brookston 8°11 C O I I O O O O O O O O O O O O O O O O O O O 79 Rates of N 0 reduction and denitrification after onset of anaerobiosis in a Flavobacterium sp. in the presence and absence of chloramphenicol . . . . 81 Trapping of 13NO in a nonlabeled NO pool in Brookston and Spinks soils . . . . . . . . . . . . 94 Influence of source composition on 13NO appearance in Brookston soil . . . . . . . . . . . . . . . . . 96 Influence of soil pH on 13NO appearance in an autoclaved and nonautoclaved Brookston soil . . . . 97 13NO production from 13N0 — in Pseudomonas fluorescens with varying concentrations of added N0 . . . . . . . . . . . . . . . . . . . . . . . . 98 13NO production and reduction in sterile and viable preparations of Pseudomonas fluorescens and Flavobacterium sp. incubated with LJ NO2 . . . 99 3N species produced from water target under our bombardment conditions. Ion distribution represents ranges encountered over many experiments . . . . . . . . . . . . . . . . . . . 109 vii Figure 10 ll 12 13 LIST OF FIGURES Exchange 95 [13N]-N 0 with added nonlabeled N20 (1.4 X 10 atm) in Pseudomonas aeruginosa . . . . 1BNH + production from 13N0 - in Pseudomonas 151 after a 10 min incubation . . . . . . . . . . . . 13NH + production from 13N0 — in a Micrococcus sp. after a 10 min incubation . . . . . . . . . . . . Exchange of [13Nl-N 0 with added nonlabeled N20 in a Brookston and a Spinks soil during a 10 min incubation . . . . . . . . . . . . . . . . . . 13N-gas production by_the Spinks soil with the addition of 0 ppm N02 -N carrier . . . . . . . . . 13N-gas production by the Spinks soil with the addition of 0.5 ppm NO2 -N carrier . . . . . . . . 13N-gas production by the Spinks soil with the addition of 4.0 ppm N02 -N carrier . . . . . . . . 13N-gas production by the Spinks soil with the addition of 8.0 ppm N02 -N carrier . . . . . . . . 13N-gas production by the Brookston soil with the addition of 20 ppm NO3 -N carrier . . . . . . . . 13N-gas production by the Brookston soil with the addition of 2.0 ppm NO3 -N carrier . . . . . . . . Influence of O on denitrification activity and products in a rookston soil . . . . . . . . . . . Influence of O on the product composition of denitrification in a Miami soil . . . . . . . . . Influence of 02 on denitrification activity in a Miami 8011 O O O O O O C O O O O O O O O O O 0 O 0 viii Page 20 24 25 27 41 42 44 45 49 50 52 55 56 Figure ‘ Page 14 Production of N 0 in a Brookston soil during early phases of incubation in the presence and absence Of C H O O O O I O O O O O O O O O O O O O O O O 68 2 2 15 Continued production of N O with time in a Brookston soil in the presence and absence of C2H2 . O I O O O O O O O O I I O O I O O O O O O 69 16 N 0 reduction and denitrification with time in a FIavobacterium sp. . . . . . . . . . . . . . . . 80 17 "Rabbit" used to contain and transport water to . the beam line C O C . . . . C C . O O . C O O O O 108 18 Diagram of gas stripping system usedlgo continuously monitor [ N]-N20 and [ N]—N2 . . . Ill 19 Electronic configuration for NaI(Tl)-PM detectors used with the gas stripping system and the well counters O O C O O O O 0 O O O O O O O O I O O O O 113 20 Diagram of gas chromatography-proportional counter (ES-PC) sysigm used to separate and quantitate [ Nl-NZ, [ Nl-NZO and N0 0 o o o o o o o o o 115 21 Typical chromatogram from GC—PC system . . . . . . 117 22 Electronic configuration employed with GC~PC SyStem O O I C O O O O O O O O C O O O O O O O O 118 23 Diagram of high pressure liquid chromatograph (HPLC) sygtem+usig to separais, quantitate, and collect NH4 , N02", and N03 . . . . . . . . 120 24 E§amp1e of separation of 13NH +, 13N0 -, and N0 by HPLC and detected by coincidence counging of annihilation gamma rays . . . . . . . 121 25 Electronic configuration employed with HPLC-NaI(T1) SYStem o o o o o o o o o o o o o o o 122 IX INTRODUCTION AND EXPERIMENTAL OBJECTIVES In the last five years there has been a renewed interest in soil denitrification due largely to three factors: the increased cost of nitrogen fertilizer; the suggestion that soil evolved nitrous oxide may contribute to a warming of the Earth's surface through a "greenhouse" effect (48); and the possible involvement of nitrous oxide in depletion of the Earth's ozone shield. The suggested effect of N O on the ozone 2 layer has received extensive publicity and generated international concern. The hypothesis is that N20, released to the atmosphere during denitrification of nitrate in soil and water, initiates photochemical reactions in the stratosphere which result in partial destruction of the ozone layer which protects the earth from biologically harmful ultra- violet radiation (14, 15, 28, 34, 35). Nitrous oxide does not directly react with ozone, but in the stratosphere it decomposes to nitric oxide which reacts with 0 to yield 02 (13, 27): 3 N20 + 0(1D) + mo [1] NO + 03 + NO2 + O2 [2] N02+O+NO+02 [3] McElroy and coworkers (34) have predicted that if denitrification rates keep pace with the increasing use of nitrogen fertilizer, the resulting reduction in total ozone would be 20% or more by the year 2025. In contrast, Liu £3 31. (31) have argued that at most a 1% reduction would occur by 2025. The difficulties in such predictions arise from unknown parameters in the atmospheric chemistry and from the uncertainty as to the magnitude of the various sources and sinks for N20 (12, 14, 24, 31, 32, 34, 45, 50). Although it is generally agreed that soil is a major source of N20 (6) it has also been suggested that soil may serve as an important sink for N20 (4, 7). Recent work by Freney and coworkers (21) indicated that under normal conditions, field soils are more likely to be net sources than sinks for atmospheric N20. Estimates of nitrogen lost due to denitrification of applied fertilizer range from 15% (l) to 70% (40). A thorough review of the denitrification literature has lead Hauck to conclude that the best estimates of average N loss from agricultural soils lie between 20 and 30% (Fertilizer Institute, Denitrification Seminar, San Francisco, CA, Oct. 25-27, 1977). The proportion of the nitrogen gas lost as N20 has been reported to vary between 0 and 100% depending on the conditions existing in the zone of denitrification (9, 11, 37, 49). But most of these estimates were determined in sealed systems which differ signifi- cantly from the field situation. The CAST Report (12) suggests that an N2 to N20 ratio of around 16 (about 6% N20) may be typical of agricul- tural soils. However, it is becoming increasingly apparent that there is no single ratio of N2/N20 which will be generally applicable to soils, for the composition of gaseous denitrification products varies tremendously with conditions in soil. Estimates of the N2/N20 gas ratio in field soils have been very difficult to obtain due to methodological limitations. The discovery and application of the acetylene inhibition technique (acetylene inhibits the reduction of N O to N2) may aid in 2 obtaining better denitrification estimates (3, 29, 42, 51, 52), but it too has inherent problems (42). This technique will be discussed further in Chapter III. Nitrous oxide is an intermediate and potential end product of the proposed denitrification pathway (39): N03 + N02" —> N0 + N20 —+ N2. The evidence supporting this pathway is extensive and has been well summarized in several excellent reviews (17, 18, 20, 39). Most deni- O to N and it has been 2 2 shown in a number of strains that this reductive step is coupled to trifying organisms are capable of reducing N energy yielding phosphorylation (2, 26, 30, 46). A recent survey of 77 denitrifying isolates by G. U. Okereke has shown that 82% can grow using N20 as the sole terminal electron acceptor (38). It thus seems somewhat inefficient for N20 to be lost as an end product of the reductive sequence. St. John and Hollocher (43) have shown that N O is a "freely 2 diffusable" intermediate in Pseudomonas aeruginosa. This was done by observing the exchange of the 15N isotOpe added as 15N02- with a pool of nonlabeled N20. The implication is, that N20 can easily escape the reductive process; that for at least a short period of time it is free to diffuse away from the site of active denitrification. If this were found to be a general characteristic of denitrifying organisms or of the mixed denitrifying flora of soil, then it would provide a basis for understanding why N20 can occur as a product of denitrification. It may also provide the mechanism by which various factors can influence the proportion of the gaseous products released as N O or N . If a factor 2 2 decreased the rate of N20 reduction without correspondingly decreasing 4 the rate of N20 production then the free N20 could diffuse away from the active site. Similarly if a factor caused an increase in the rate of N20 production without effecting a comparable increase in the rate of N20 reduction, then N20 could again be lost from the reductive process. A number of early investigators of denitrification in soils observed that specific environmental conditions seemed to enhance the production of N20 relative to N2 during denitrification. The factor that was first recognized to influence the composition of the gaseous products of denitrification was pH. In 1954, Wijler and Delwiche (49) reported that N20 was the major product of denitrification in acid soils while at neutral or alkaline pH's, N2 was the dominant product. A number of other investigators have reported similar findings (8, 25, 33, 37). Recently, Blackmer and Bremner (5), have suggested that the pH effect on the N20/N2 ratio is actually the influence of N03- concentration, which is strongly enhanced in acid soils. The N20 reducing activity of the low pH soils studied by these investigators, was actUally higher than that of neutral and alkaline soils. Yet, in the presence of high concentrations of N03-, more N20 accumulated in the acid soils than in the neutral or alkaline soils (5). The availability of carbon has been reported to influence the extent to which denitrification goes to completion. Wijler and Delwiche (49) and Nommik (37) found that when exogenous carbonaceous substrate was added to soils, proportionally less N20 was evolved. Stefanson (44) and Smith (41) have reported that the N20/N2 ratio of denitrification is lower in soils of the plant rhizosphere than in unplanted soils. The recent work by Smith has indicated that this effect may be due to increased carbon availability rather than to reduced 0 tension (41). 2 It is quite well known that 0 strongly influences denitrification. 2 Increasing partial pressures of 02 in soils result in decreases in denitrification activity. The kinetic model proposed by Focht (19), based largely on Nommik's early work (37), predicted that the aeration status of soils should also influence the composition of the gaseous products of denitrification. This model, and work by Cady and Bartholomew (10), indicate that as the availability of 0 increases, the N20/N2 2 ratio also increases. It is possible that the effect of 02 on the ratio of N20 to N2 produced results from the influence of O on the redox 2 potential of the soil environment. In his review: Focht calculates a redox couple (Eh) for N20/N2 in aqueous solutions of 250 mV (20). Hence, N20 may be more likely to accumulate when the redox potential does not fall below this value. Several of the early investigators observed that high N03- concen- trations resulted in elevated proportions of N20 as the product of denitrification (10, ll, 37, 49). Although it has been suggested that N03- was preferentially used over N20 as an electron acceptor (10, 16, 36, 39), there was no direct evidence that this was indeed the mechanism. Blackmer and Bremner have recently reported that N03 concentrations of 10 ppm NO3-N and above result in higher prOportions of N O as the 2 terminal product (5). The factors which control the composition of the gaseous products of denitrification which escape from soil to the atmosphere, can be classified into two categories.' First, there are those factors which control the biological release of N20. These parameters act at a cellular level and can result in N20 which is transiently free from the reductive process. The second category of factors Operates at a soil structural level and controls the ease with which N20 can escape the active microbial community. This group includes soil porosity, water content, texture, and any characteristic which influences the rate of diffusion. In many instances there is a strong interaction between the two levels of control. For example the influence of 02 would be exerted at a cellular level but its presence is controlled by the O diffusion 2 characteristics of the soil. It is apparent that for our knowledge to have predictive value, we must understand the control exerted at both levels. But a myriad of interacting and constantly changing parameters control the composition of the gases evolved from soil. Hence it seemed that a first step should be an understanding of the biological control of N20 production in soil. Most of my research has been focused on factors which control the biological release of N O in soils. This work has included the use of 2 bacterial cultures and soil slurries (in which the effects of soil structure are minimized). The bacterial strains used were chosen as representative of the numerically dominant denitrifiers isolated from several soils (22). The experimental techniques employed are relatively new. I have used the longest—lived radioactive isotOpe of nitrogen, 13N (half-life 9.96 min) for much of my research. In 1976 Gersberg £5 31. (23) reported on the use of 13NO3- in studies to determine the natural rates of denitrification in sediments. In the last two years we have developed and applied 13N techniques to a number of aspects of denitri- fication research. A brief introduction to these procedures has been published (47). The use of the 13N isotOpe provides an extremely sensitive, direct approach for short-term denitrification studies. A description of the techniques employed is included in the Appendix to this thesis. The second experimental approach used in this research involves the use of the acetylene inhibition technique. In 1976 Yoshinari and Knowles (51) and Balderston g£_§1, (3) reported that acetylene inhibits the reduction of N20 to N2. In the presence of sufficient acetylene and N03-, N20 is the only gaseous product of denitrification (3, 29, 42, 51, 52). Nitrous oxide can be easily and sensitively quantified by use of a gas chromatograph equipped with a thermal conductivity detector, for concentrations above 50 ppm, or an electron capture detector, for concentrations near or below ambient (296 ppb). This procedure eliminates the major problem which has hindered research in denitrification, the inability to directly measure the production of small quantities of N2 in a natural atmosphere containing 79% N2. Even in artificial atmo- spheres from which N2 has been removed, contamination by atmospheric N2 during sampling and analysis has posed a difficult problem. A more complete explanation of the acetylene inhibition technique is included in Chapter III. My research centered on the following questions: 1. Is N20 free to diffuse away from the active site of denitrifi- cation? Environmental conditions can thus influence whether N O is lost or reduced to N . 2 2 2. Does the availability of other electron acceptors (NO —, N02- and 02) influence the prOportion of N 0 and N2 produced by 2 denitrification? Does the ratio of N20 to N2 change with time after the initiation of denitrification by the onset of anaerobic conditions? Does the availability of carbon affect the composition of the gaseous products of denitrification? Is nitric oxide, or a compound with which N0 is in equilibrium, an obligatory intermediate in denitrification? 10. 11. LITERATURE CITED Allison, F. E. 1955. The enigma of soil nitrogen balance sheets. Adv. Agron. 7:213—250. Baldensperger, J. and J. L. Garcia. 1975. Reduction of oxidized nitrogen compounds by a new strain of Thiobacillus denitrificans. Arch. Mikrobiol. 103:31-36. Balderston, W. L., B. Sherr, and W. J. Payne. 1976. Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus. Appl. Environ. 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The use of acetylene inhibition of nitrous oxide reductase in quantifying denitrification in soils. Swedish J. of Agricultural Res. 7:179. Koike, I. and A. Hattori. .1975. Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseudomonas denitrificans under nitrate-, nitrite-, and nitrous oxide-limited conditions. J. Gen. Microbiol. 88:11-19. Liu, S. C., R. J. Cicerone, T. M. Donahue and W. L. Chameides. 1976. Limitation of fertilizer induced ozone reduction by the long lifetime of the reservoir of fixed nitrogen. GeOphys. Res. Lett. 3:157-160. Liu, S. C., R. J. Cicerone, T. M. Donahue and W. L. Chameides. 1977. Sources and sinks of atmospheric N O and the possible ozone reduction due to industrial fixed nitrogen fertilizers. Tellus 29:251-263. Matsubara, T. and T. Mori. 1968. Studies on denitrification. IX. Nitrous oxide, its production and reduction to nitrogen. J. Biochem. 64:863-871. McElroy, M. B., J. W. Elkins, S. C. Wofsy and Y. L. Yung. 1976. Sources and sinks for atmospheric N20. Rev. GeOphys. Space Phys. 14:143-150. McElroy, M. B., S. C. Wofsy and Y. L. Yung. 1977. The nitrogen cycle: perturbations due to man and their impact on atmospheric N20 and 03. Phil. Trans. R. Soc. 277B:159-181. Myers, R. J. K. and J. W. McGarity. 1972. Denitrification in undistrubed cores from a solodized solonetz B horizon. Plant and Soil 37:81-89. Nommik, H. 1956. Investigations on denitrificaton in soil. Acta Agr. Scand. 6:195-228. Okereke, G. U. 1978. Utilization and production of N 0 by denitri- fiers isolated from different soil environments and effect of pH on the rates and products of denitrification. M.S. Thesis, Michigan State University. Payne, W. J. 1973. Reduction of nitrogenous oxides by micro- organisms. Bacteriol. Rev. 37:409-452. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 12 Rolstan, D. E., M. Fried and D. A. Goldhamer. 1976. Denitrifica- tion measured directly from nitrogen and nitrous oxide gas fluxes. Soil Sci. Soc. Amer. J. 40:259-266. Smith, M. S. 1978. Short-term measurement of soil denitrification using C H inhibition: response to anaerobiosis and the effect of the rhizosphere. Ph.D. Thesis, Michigan State University. Univer- sity Microfilms, Ann Arbor, MI. Smith, M. S., M. K. Firestone and J. M. Tiedje. 1978. The acetylene inhibition method for short-term measurement of soil denitrification and its evaluation using nitrogen-l3. Soil Sci. Soc. of Am. J. 42:611-615. St. John, R. T. and T. C. Hollocher. 1977. Nitrogen 15 tracer studies on the pathway of denitrification in Pseudomonas aeruginosa. Stefanson, R. C. 1972. Soil denitrification in sealed soil-plant systems. I. Effect of plants, soil water content and soil organic matter content. Plant and Soil 37:113-127. Sze, N. D., and H. Rice. 1976. Nitrogen cycle factors contributing to N20 production from fertilizers. GeOphys. Res. Lett. 3:343-346. Terai H. and T. Mori. 1975. Studies on phosphorylation coupled with denitrification and aerobic respiration in Pseudomonas denitrificans. Bot. Mag. (Tokyo) 88:231-244. Tiedje, J. M., M. S. Smith, M. K. Firestone, M. R. Betlach and R. B. Firestone. 1977. hart term measurement of denitrification rates in soils using N and acetylene inhibition methods. In Proceedings International Symposium on Microbial Ecology, Dunedin, NZ, Aug. 1977. Springer-Verlag (in press). Wang, W. C., Y. L. Yung, A. L. Lacis, T. Mo and J. E. Hanson. 1976. Greenhouse effects due to man-made perturbations of trace gases. Science. 1974:685-689. Wijler, J. and C. C. Delwiche. 1954. Investigations on the deni- trifying process in soil. Plant and Soil 5:155-169. Yoshinari, T. 1976. Nitrous oxide in the sea. Marine Chemistry 4:189-202. Yoshinari, T. and R. Knowles. 1976. Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria. Biochem. Biophys. Res. Comm. 69:705-710. Yoshinari, T., R. Hynes and R. Knowles. 1977. Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biol. Biochem. 9:177—183. CHAPTER I STUDIES ON NITROUS OXIDE AS A FREE, DIFFUSABLE INTERMEDIATE OF DENITRIFICATION IN SOILS AND BACTERIAL CULTURES It is generally accepted that N20 is an intermediate in denitrifi- cation; the evidence supporting this is extensive and has been well summarized in several recent reviews (5, 8, 14). It is commonly observed in soils and in bacterial cultures that denitrification can result in N20 as well as N2 as a terminal product. However, the loss of N20 from the reductive sequence may represent an energy loss to the denitrifying microflora. Delwiche, in his paper on the thermodynamics of N20 evolution (6), concluded that "the appearance of N20 in denitrification must reflect a metabolic accident or some reaction barrier . . . which prevents the full utilization of this energy." The first step in understanding why N20 is lost from the denitrification process, is to determine if N20 generally exists as a free intermediate, capable of diffusion away from the active site of reduction. St. John and Hallocher (l6), studying the reduction of 15NO _ in 2 Pseudomonas aeruginosa, concluded that N 0 is a free, obligatory inter- 2 mediate in the reduction of N02- to N2. The purpose of my work was to determine whether N20 is generally a "free' and "obligatory" inter- mediate in the heterogeneous denitrifying microflora of soils. To do this I added 13N-labeled N03- to soils and pure cultures. The N20 intermediate of denitrification would thus be 13N labeled. I then quantified the mixing or exchange of the [13Nl-N20 with pools of added, nonlabeled N20. 13 14 Several years ago, two groups of researchers, Yoshinari and Knowles (18) and Balderston_et‘al. (1) reported that acetylene inhibited the reduction of N20 to N2 in pure cultures and in soils. With the addition of sufficient acetylene and N03 , the blockage of N 0 reduction was 2 complete and resulted in stoichiometric accumulations of N20 (1, 15, 18, 19). This has been interpreted as strong confirmatory evidence for the obligatory nature of N 0 in the reductive sequence of denitrification. 2 Yoshinari and Knowles (19) reported that the kinetic characteristics of the acetylene effector were that of a noncompetitive inhibitor, but generally very little is known about the mechanism of inhibition. Recently, Focht, £5 £1. (8) reported isolation of two denitrifying bacteria, an Alcaligenes sp. and a Micrococcus sp., that did not produce N20 in the presence of acetylene. These organisms were also incapable of reducing added N20 to N2. Focht sent me these cultures with the hope that I could determine whether or not N20 existed as an intermediate in the reductive sequence. Gilbert Okereke, in our laboratory, had also determined that one of Gamble's isolates (9), Pseudomonas strain 151, did not produce N20 in the presence of acetylene and could not grow on N20 (13). In this chapter I report the results of [13N]-N20 exchange studies with these three isolates, three "normal" denitrifying strains and two soils. MATERIALS AND METHODS Bacterial isolates and cultivation Four of the bacterial strains used in this study were isolated by T. N. Gamble from soil samples obtained from various parts of the world. 15 These organisms were fully characterized during earlier work in our laboratory (9, 10). These organisms were: Pseudomonas fluorescens biotype II strain 72, Flavobacterium sp. strain 175, Pseudomonas aeruginasa strain 156, and Pseudomonas sp. strain 151. The two other strains used were a Micrococcus sp. and an Alcaligenes sp. obtained from D. D. Focht, University of California, Riverside. The bacterial isolates from our laboratory were grown in 2 l of Nitrate Broth (Difco Laboratories, Detroit, MI) under anaerobic condi- tions in a Freter glove box (Coy Manufacturing, Ann Arbor, MI). The two strains from Focht were grown in 2 l Tryptic Soy Broth (Difco Laboratories) plus 3.5 mM KNO3 and 0.1% sodium succinate, under anaerobic conditions. Satisfactory growth of these two organisms could not be obtained under anaerobic conditions in our laboratory using Nitrate Broth. Tryptic Soy Broth with N03- and succinate was found to produce the best growth. But even in this medium, growth of the Micrococcus and Alcaligenes was poor and unreliable. After about 24 hr of growth, cells were harvested by centrifuging at 2000 g for 15 min at 2° C. The cells were resuspended in 200 ml of 0.02 M phosphate buffer, pH 7.2. This procedure was repeated three times to remove residual N03- and nutrients. The cells were then resuspended to the desired volumes; cell densities ranged from 5.0 to 8.7 mg dry weight/m1. Five milliter quantities of the cell suspensions were added to 27-ml serum vials containing 1 ml of a 1% glucose solution, giving a final glucose concentration of 0.17%. The vials were then capped with silicon septa and aluminum crimp seals. The serum vials were evacuated and filled with helium three times and stared at 4° C until used. 16 Soils used and preparation The soils used were a Brookston loam and a Spinks loamy sand described in Table 6 of Chapter II. Fifty gram fresh weight of soil and 40 ml of H20 were added to 125-ml Erlenmeyer flasks which were sealed with rubber stappers, pierced by a glass tube capped with a serum stapper. The soil slurries were made anaerobic by evacuating and filling with helium three times and then incubated for 2 days at 28° C prior to experimentation. Previous work had indicated that this 48 hr anaerobic preincubation was sufficient to deplete the naturally occurring quantities of NO3- and N02- in the two soils used. I had also found that the N20 reducing activity of these soils was high after 48 hours of anaerobiosis (Chapter III) and hence this provided ideal conditions under which to test exchange of the N20 intermediate with an external N20 pool. 13N production and experimentation 13M labeled NO3- and N02- were produced at the MSU Cyclotron by a 160(p, a) 13N reaction of a proton beam with a 0.85 ml H20 target. This procedure is described in greater detail in the Appendix. The 13N species produced were predominantly 13N03- (75-90%) with smaller quanti- ties of 13N02- and 13NH“+ also present in the solution. The 13NH4+ was removed prior to use by addition of 0.1 ml of 10 mM NaOH to the irradiated water and evacuating to dryness under a vacuum at 70° C. The sample was neutralized by the addition of 0.1 ml of 10 mM HCl and taken to the apprOpriate volume with water (1.3 or 2.5 ml for pure culture, 4.0 ml for soils). Approximately 2 min prior to the addition of the 13N substrate, the desired quantities of nonlabeled N20 were injected into the culture 17 vials or soil flasks. These were returned to a rotary shaker and agitated at 250 rpm for 2 min. The 13N substrate (0.5 ml for cultures; 1.0 ml for soils) was then injected. Nonlabeled N03- carrier was not added in these experiments. After the specified times of incubation (2- 30 min), a sample was removed from the headspace gas of each (0.5 ml for cultures; 2.0 ml for soils) using l-ml syringes (5-ml syringes for soils) fitted with Gas-Tight valves (Anspec Co., Ann Arbor, MI). The relative quantities of [13Nl-N20 and [13Nl-N2 in the gas samples were determined using the gas chromatograph-proportional counter apparatus described and pictured in the Appendix (Figure 20). Data were collected and stored using a Sigma-7 computer which was later used for off-line background and half-life corrections and peak integrations. For each experiment (of about 30-min duration), all of the collected data were corrected for half-life to a single zero time which was about 2 min before the first GC injection. The data were accumulated as disintegrations per 2 sec intervals by a Sigma-7 computer. The computer program used for data analysis integrated the number of disintegrations detected during the approximately 20 sec residence time of the gas peak in the proportional counter. The efficiency of the proportional counter approached 100%. Thus the [13N]-gas values are expressed as disintegrations, not dps or dpm. Although these units are rather unorthodox to biologists, they have the advantage of allowing the reader to evaluate directly the accuracy of the data. When the data are presented as disintegrations the numbers are followed by an estimate of the standard deviation (of the peak area) derived using a non-linear least-squares fitting procedure. (Appendix) 18 In addition to analysis of the headspace gas, the 13N ionic species in the aqueous phase were also determined for the Micrococcus sp., Alcaligenes sp. and Pseudomonas sp. After the gas samples were taken, the culture suspensions were filtered through a 0.45 u Metricel filter (Gelman Co., Ann Arbor, MI). A known volume of the filtrate was then evaporated to dryness and resuspended with 300 pl of H20. A lOO-ul subsample was injected into a high pressure liquid chromatograph contain- ing a Partisil 10/25 SAX anion exchange column (Whatman Inc., Clifton, NJ) described and pictured in the Appendix (Figure 23). The column effluent passed through a coil adjacent to two NaI(Tl) detectors for quantitation of 13NH4+, 13N03— and 13N02”. The data were recorded and analyzed using the Sigma-7 computer in a procedure essentially identical to that for the gas chromatograph-praportional counter system. RESULTS Pure cultures During denitrification of 13N03-, a l3N-labeled N20 intermediate should occur. The exchange of this [13N]-N20 intermediate with a nonlabeled pool of added N20 in three bacterial cultures is shown in Table l and Figure 1. In the absence of added nonlabeled N20, 3; fluorescens produced almost entirely [13N]-N2 (Table 1). When a nonlabeled N20 pool was added (3.0 x 10-2 atm), the 13N label appeared predominantly as [13 N]-N20. At 2 min, 93% of the 13N-gas was as [13N]-N20 while at 12 min this was reduced to 78%. This decrease apparently resulted from partial reduction of the total N20 pool to N2 since the mass peaks of N20 decreased (as determined by a microthermistor detector). Similar results are also reported for Flavobacterium sp. (Table 1). Figure l l9 .mooaumfi>op pumpomum man mum mommzuamumo ow moon> o .HmH> you mHHoo mo unwaoz map ma «q ".mm Eafiuouomnopoflm .Hmw> pom mHHao mo unwwos who we on “homomouoaam 4M n .xooe new you poumuwmuow woodm> m k.~w Am.qao o.maom Ao.oo o.amk o.m NH m.a Ao.av m.m¢ AS.HHV ~.ooam o NH o.ooa Aa.ov q.wm Aq.ov o o.m N H.ma xk.ao m.oou Ao.mV m.o~q o N n.am aaanmuumno>maa a.~a Am.oav m.maom no.mv m.mmm o.m NH o Aq.ov o.o 1N.HHV m.ua~e o NH m.mm Am.AV A.~h~m Ao.~v m.mm~ o.m N o A~.ov o vo.ov m.m~ o N nmawommuoaam.mw cmz 3 #3538335 Nos- can «.3 35 . 833 mm» zma mo N o~znmzmHH NzqumH_ suave oNz maaamm mo mafia .Hoon o~z panonmaco: o nufis .am aafiuouamno>mflm pom mooomouapam wmooaovaomm an vmaaooua o~znmzma_ mo owoonoxm .H manna IOO 80 CD CD ES ‘CD °/. OF ”N GAS As N20 20 Figure l. 0 IO 20 EXCHANGE OF '3N20 WITH ADDED l“N20 IN PSEUDOMONAS AERUGINOSA __ O o 3 mm t— I I J l 340 5.0 N20 ADDED ( Io-ZATM) Exchange of [13Nl-N20 with added nonlabeled N20 (1.4 X 10— l 2 atm) in Pseudomonas aeruginosa. 21 shows the results of increasing additions of nonlabeled N20 to 2;. aeruginosa. As the size of the nonlabeled N20 pool was increased more of the [13Nl-N20 was exchanged and trapped in the N 20 pool. Again the percentage of the label trapped in the N 0 pool decreased with time from 2 the 3 to 30 min samples. In these three denitrifying cultures, the N20 intermediate readily mixed or exchanged with externally added N20. Similar experiments were performed with the three cultures which did not produce N20 in the presence of acetylene with rather surprising results. Essentially no [13N]-N20 or [13N]-N2 was produced (Table 2). 13 Very little (<0.08%) of the added N03- was reduced to gaseous products. Analysis of the aqueous phase of the incubations showed that Pseudomonas strain 151 reduced a significant portion of the 13NO - to 13NH4+ 3 13 - 13 + (Table 3). A slight reduction of the N03 to. NH4 by the Micrococcus may also be indicated by these data. 13N-chramatograms from the HPLC- NaI(Tl) system for these two cultures are shown in Figures 2 and 3. The increases in 13NH,‘+ in the samples incubated with Pseudomonas 151 and Micrococcus are reasonably apparent. Unfortunately, I had some difficulty growing the Alcaligenes and Micrococcus for this experiment. For a previous cyclotron experiment I had managed to obtain good growth of the Alcaligenes. A similar experiment performed with Alcaligenes at that time indicated conversion of 42% of the 13N03- to 13NH4+. However, the concentration of the filtrate was not done quantitatively and hence I could not conclude that the total percentage increase in 13NH4+ in the filtrate resulted from production of 13N114+ rather than from an' unexplained disappearance of 13N03- and 13N02-. 22 Table 2. l3N-gas production by Pseudomonas 151, Alcaligenes sp. and Micrococcus sp. [13N]-N2a [13N]-N20a TotallBN-gasa % of total Culture ----------(disintegrations) 13M addedb Pseudomonas 151 112 (17)c 66 (15) 178 0.08 Alcaligenes sp. 0 0 0 0 Micrococcus sp. 88 (25) 0 88 0.08 a Gas samples were taken after a 10 min incubation. b Total 1:8 added was calculated from HPLC analysis of a 50 ul sample of the N added. Buffer flow through the HPLC was 2 ml/min and the detector efficiency was approximately 10%. Gas flow through the GC was 63.5 ml/min; the PC volume was 25 m1 and the detector efficiency was 100%. c Values in parenthesis are the standard deviations. 23 Table 3. 13N ionic species found after incubation of 13N substrate with Pseudomonas 151, Alcaligenes sp. and Micrococcus sp. a a ---;A of Total activity added -:- % of Total Treatment NH4 N02 N03 recovered Untreated Ab 0.1 (.02)C 9.4 (.14) 90.5 (.45) 100.0 Pseudomonas 151 9.2 (.18) 13.2 (.21) 64.1 (.49) 86.5 Alcaligenes sp. 0.3 (.11) 9.0 (.35) 98.1 (1.15) 107.4 Untreated B 0.0 (.02) 14.5 (.25) 85.5 (.62) 100.0 Micrococcus sp. 1.7 (.18) 29.4 (.59) 106.2 (1.15) 137.3 a Total activity added to Pseudomonas 151 and Alcaligenes was 10.4 nCi; Micrococcus - 5.2 uCi. The cultures were filtered after a 10 min incubation. b Untreated A shows the composition of the 13N substrate added to Pseudomonas 151 and Alcaligenes; Untreated B to Micrococcus. c Values in parentheses are the standard deviations. 24 “N03- 1) ”a? Z O H . t;E -. I13rq(:l2" ‘- m 8 .2 |3NH4+ PS 5; _. TYPE 19 ~ f—{ 9.. 27‘ u w. .1 k UNTREATED % : ¥+ 2 4 8 TIME [MIN] after a 10 min incubation. 25 "NO.“ ) "NO." L p d M-COCCUS "N (DISINTEGRATIONS) f\ UNTREATED é Li 6 TIME [MIN] Figure 3. NH4+ production from 13N0 - in a Micrococcus sp. after 3 a 10 min incubation. 26 mug The exchange of the [13Nl-N20 intermediate with added N20 pools in two soils is shown in Figure 4 and Tables 4 and 5. From Figure 4, it can be seen that as the size of the nonlabeled N20 pool was increased, the prOportion of 53N trapped as N20 increased. It is apparent from the data in Tables 4 and 5 that the addition of even the smallest quantity of N20 (1.2 x 10.2 atm) resulted in almost complete trapping of the 13 [13N]-N20 in the N 0 pool (about 99% of the N label). The sterile 2 soil control shown at the bottom of Table 4, indicates that no non- biological production of [13N]-N20 or [13Nl-N occurred. The data 2 indicate that the N20 intermediate of denitrification in these soils readily exchanged with an added N 0 pool. 2 DISCUSSION When 13N03- was added to a Brookston and a Spinks soil, the 13N was trapped almost quantitatively as [13Nl-N20 in a pool of added N20. The make-up of the denitrifying microflora in soils is known to be heteroge- neous (9, 10). Thus for the diverse populations active in soil denitrifi- cation, the labeled N20 intermediate mixed rapidly and quantitatively with an added pool of N20. This supports the role of N20 as an obligatory intermediate of denitrification in soils. The freedom with which the N20 intermediate exchanged with an added N20 pool indicates that Operation- ally, N20 exists as a free intermediate able to diffuse away from the active site of reduction. This then may provide the basis of understand- ing why, under some conditions, N20 occurs as the product of denitrifica- tion in soils. N20/ N2 27 EXCHANGE OF '3N20 me ADDED ”N20 500 l 400 - Ema/r5 Ion 320/7715 I 200 IOO I 1 1 1 l 1 1 1 1 4 8 :2 IE ”N20 ADDED ( :02 ATM) Figure 4. Exchange of [13Nl-N20 with added nonlabeled N20 in a Brookston and a Spinks soil during a 10 min incubation. 28 Table 4. Exchange of [13Nl-N20 produced by a Brookston soil with a nonlabeled N20 pool. Time of sample N20 added [13Nl-N2 13N-N20 13 % of 13N (min) (10"2 atm) (102 disintegrations)a [ Nl-NZO/NZ gas as N20 10 0 4083 (.64)b 68 (.09) 0.02 1.6 1.4 16 (.05) 1185 (.35) 74.3 98.7 7.0 C 2 (.02) 686 (.26) 295. 99.7 14.0 5 (.04) 2377 (.49) 443. 99.8 28.0 4 (.03) 1970 (.45) 492. 99.8 30 0 4361 (.66) 16 (.10) 0.00 0.4 1.4 58 (.81) 2524 (.50) 43.6 97.8 7.0 c 15 (.05) 1633 (.41) 108. 99.1 14.0 11 (.05) 2318 (.48) 220. 99.6 28.0 1 (.07) 2245 (.48) 3164. 100.0 26 (autoclaved) 0 0 0 - - 7.0 O 0 - - a Values integrated for gas peak. Values in parentheses are the standard deviation. c Indicates different experiments and thus different amounts of N substrate added. 29 .mo08omfi>av oumocmum mum momosuooume o8 moaam> n .mxmon mow uo>o ooumumoBoH mosam> m 8.88 .an Am8.8 ~8~H a8o.o ma 8.8a 8.88 H.8m Ane.v mmHN a8o.8 mm «.8 “.88 o.- AH8.8 88m~ Aaa.v 888 ~.H 8.8 88.8 Ana.v 88 Ama.v 8888 8 on A.88 .888 A88.8 mama Amo.8 m 8.88 8.88 .mHN A88.8 mmma a8o.8 8 N.8 8.88 «.88 An8.8 8-~ Amo.v 88 ~.H 8.88 AH.8 An~.v 888 8a88.8 8NH8 8 8H omz mm Nz\ONZIHZma_ IEAMGOAuoquuMHmfio NOHvI Aaum NIMHV oaaaomhwwawafia 888 zmH 88 N o zu_zma_ zTHZmH_ 88888 o z 8 s8H3 8888 .Hooa coaonoaooa Nzu_zma_ 88 mmamnuxm .8 88888 oxafiam m mp couscoua o 30 In the three denitrifying cultures examined, 2; fluorescens, Flavobacterium sp. and P;_aeruginosa, 13N from 13NO3- was also trapped 0 pool. Nitrous oxide almost completely as [13Nl-N20 in a nonlabeled N2 appears to be an Operationally free, obligate intermediate in the reduc- tion of N03- to N2. The facility with which the [13N]-N20 intermediate exchanged with the added N20 pool suggests that N20 must readily move through the cell envelope of these bacteria or that it exists at least transiently as an intermediate external to the cell envelope. The possibility that N20 may exist as an external intermediate is quite interesting in light of previously reported work with respiratory nitrate reductase of Escherichia coli. Garland gt a1. (11) and Boxer and Clegg (2) have concluded that nitrate is reduced to nitrite on the outer aspect of the E; £211 membrane and that nitrate never actually enters the cell. Nitrous oxide reductase has been reported to be a membrane bound enzyme (4), but its orientation with respect to the membrane is not known. The most surprising result of this work was the production of IBNH4+ from 13NO3- by Pseudomonas 151, Alcaligenes and possibly by Micrococcus. I have observed that the growth characteristics of these three strains, under anaerobic conditions in the presence of N03-, are quite different than those of the other denitrifying cultures with which I have worked. These organisms grew more slowly and to a lower final density than those cultures which are known to reduce N03 to N2. This may be consistent with a different scheme of NO3 reduction in these organisms. 31 Dissimilatory reduction of N03— to N114+ has been reported in a number of organisms: strict anaerobes (3), enteric bacteria (17) and several Bacillus species (12). Only in the enteric bacteria, is the reduction of N03- directly coupled to respiratory electron transport. Oxidative phosphorylation results only from the reduction of N03- to N02- (17). I have no evidence indicating that in the cultures used in this study, the reduction of N03- to NH4+ is coupled to respiration or that it is truly dissimilatory. It should be apparent that the data reported here on N03- reduction to NH4+ represent the beginning of an investigation not the conclusion. The Pseudomonas 151, Micrococcus and Alcaligenes cultures were chosen for study because they did not produce N20 in the presence of acetylene. These seemed to be good strains in which to test the hypoth- esis that N20 is generally a free intermediate of denitrification. I had assumed that the only reductive fate of N03- in these strains was the gaseous denitrification products. It is possible that denitrifica- tion of N03- to N2 and anaerobic reduction of N03- to NH4+ occurs in the same organism and that the conditions which I employed favored the reduction of N03- to NH4+. The characteristic on which these cultures were chosen (absence of N20 accumulation in the presence of CZHZ) is apparently not a common one. In a survey of 77 "denitrifying" cultures, G. Okereke found only two strains which did not accumulate N 0 in the 2 presence of acetylene (13). Three reductive sequences involving N20 have been considered as possible pathways for denitrification: 32 [1] N02- > X > N2 ‘7 \A / N20 [2] N02 > 35 > N2 V N20 [3] N02 ———9 N20 > N2 1| V N20 In the first sequence, two pathways of reduction are possible, both yielding N2 as the final product, but only one involving N20. The data presented here can be interpreted as strong evidence against such a pathway. The 13N-label from 13NO3- was trapped almost quantitatively as [13N]-N20 in a nonlabeled N20 pool. As the size of the nonlabeled pool was increased, the quantity of 13N-label trapped in the N20 pool increased. The second sequence involves the production of an intermediate with which N20 exists in equilibrium. Nitroxyl (or its dimer HON:NOH) has been suggested as such an intermediate. The data presented here cannot rule out this sequence. However, this pathway requires the existence of a species with which N20 can rapidly equilibrate. There are nitrogen oxides which can spontaneously decompose to farm N20, but the reverse step of the equilibrium, the spontaneous conversion of N 0 to an X 2 intermediate is highly unlikely. Nitrous oxide is a stable chemical species. The third sequence shows N20 as an obligatory intermediate in the pathway to N2. I believe that the data reported here strongly support the hypothesis that N20 is generally a free and obligatory intermediate of denitrification. 10. 11. LITERATURE CITED Balderston, W. L., B. Sherr, and W. J. Payne. 1976. Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus. Appl. Environ. Microbiol. 31:504-508. Boxer, D. H. and R. A. Clegg. 1975. A transmembrane location for the proton translocating reduced ubiquinone - nitrate reductase segment of the respiratory chain of Escherichia coli. FEBS Letts. Caskey, W. H. 1978. Studies on dissimilatory reduction of nitrate to ammonium by soil Clostridium spp. Ph.D. Thesis. Michigan State University. University Microfilms, Ann Arbor, Mich. Cox, C. D. and W. J. Payne. 1973. Separation of soluble deni- trifying enzymes and cytochromes from Pseudomonas perfectomarinus. Can. J. Microbiol. 19:861-872. Delwiche, C. D. and B. A. Bryan. 1976. Denitrification. Ann. Rev. Microbiol. 30:241-262. Delwiche, C. C. 1978. Biological production and utilization of N20. PageOph. 116:414-424. Focht, D. D. and W. Verstraete. 1971. Biochemical ecology of nitrification and denitrification. Pages 135-214 ig_M. Alexander, ed. Advances in Microbial Ecology, vol. 1. Plenum Press. Focht, D. C., H. Joseph and R. Zablotowicz. 1978. Comparison of dissimilatory nitrate and nitrous oxide reduction among several genera of soil bacteria. Abstr. Annu. Meet. Am. Soc. Microbial. Q 14. p. 197. Gamble, T. N. 1976. The commonality of numerically dominant denitrifier strains isolated from various habitats. M. S. Thesis, Michigan State University. Gamble, T. N., M. R. Betlach and J. M. Tiedje. 1977. Numerically dominant denitrifying bacteria from world soils. Appl. Environ. Microbiol. 33:926-939. Garland, P. B., J. A. Downie and B. A. Haddock. 1975. Proton translocation and the respiratory nitrate reductase of Escherichia coli. Biochem. J. 152:547-559. 33 12. l3. 14. 15. 16. 17. 18. 19. 34 Ilina, T. K., and R. N. Khodakova. 1976. Chemistry of denitrifica- tion in sporeforming sail bacteria. Mikrobiologya 45:602-606. Okereke, G. U. 1978. Utilization and production of N O by deni- trifiers isolated from different soil environments and effect of pH on the rates and products of denitrification. M.S. Thesis, Michigan State University. Payne, W. J. 1973. Reduction of nitrogenous oxides by micro- organisms. Bacteriol. Rev. 37:409-452. Smith, M. S., M. K. Firestone and J. M. Tiedje. 1978. The acetylene inhibition method for short-term measurement of soil denitrification and its evaluation using nitrogen-13. Soil Sci. Soc. Am. J. 42: 611-615. St. John, R. T. and T. C. Hollocher. 1977. Nitrogen 15 tracer studies on the pathway of denitrification in Pseudomonas aeruginosa. J. Biol. Chem. 252:212-217. . Thauer, R. K., K. Jungermann and K. Decker. 1977. Energy conserva- tion in chemotrophic anaerobic bacteria. Bacterial. Rev. 41:100- 180. Yoshinari, T. and R. Knowles. 1976. Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria. Biochem. BIOphys. Yoshinari, T., R. Hynes and R. Knowles. 1977. Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biol. Biochem. 9:177-183. 35 CHAPTER II THE INFLUENCE OF NITRATE, NITRITE, AND OXYGEN CONCENTRATIONS ON THE COMPOSITION OF THE GASEOUS PRODUCTS OF DENITRIFICATION IN SOIL The environmental factors which control denitrification are generally well known and have been discussed in several reviews (7, 8, 11). It is now widely accepted that N20 and N are common end products of deni— 2 trification in soils; but the production of N20 relative to N2 varies significantly in different soils and in the same soil under different conditions. The parameters which control the production of N20 relative to N2 are not well understood. A number of investigators have reported an influence of N03— concentration and 02 status on the ratio of the product gases (1, 3, 4, 5, 6, 9, 12). But it can be difficult to define the effect of a single parameter when a number of changing or noncon- trolled parameters influence the result. In a natural soil system, one might expect N0 concentration, NO2 concentration, 02 concentration, 3 carbon availability, pH, temperature, as well as the composition and physiological state of the microbial community, to influence the composi- tion of the gaseous products of denitrification. Imposed upon the biological control of the NZO/N2 ratio are the physical characteristics of the soil--porosity, water content and texture--which control the ease with which the biologically produced N20 is lost from the soil or is available for further reduction to N2. I have used 13N-labeled N03- to investigate the influence of N0 -, N02- and 02 concentrations on the composition of the gaseous products of denitrification. Using short-term incubations, I have been able to 36 control or define the possible influential factors. To focus on the biological process I have worked in well mixed soil slurries, thus minimizing the influence of soil structure on the results. MATERIALS AND METHODS Soils used and preparation The soils used were a Brookston loam, a Spinks loamy sand and a Miami sandy loam; they are described in Table 6. After collection the moist soils were passed through a 5 mm sieve and stored in sealed plastic bags at 2° C until used. For experiments on the effect of N03- and N02- concentration, 75 g (fresh weight) of soil and 50 m1 H20 were added to 125-m1 Erlenmeyer flasks which were sealed with rubber stoppers, pierced by a glass tube capped with a serum stapper. The soil slurries were made anaerobic by evacuating and filling with helium three times and then incubated at 28° C for two days prior to experimentation. Previous work had indicated that this 48-hour anaerobic preincubation was sufficient to deplete the - and NO - in the soils used. I had 3 2 also found that the N20 reducing activity of these soils was high at 48 hours (Chapter III) and hence preincubated soils provided the most naturally occuring quantities of NO rigorous conditions under which to test factors which could cause N20 accumulation. The same preincubation procedure was used for experiments with 02. This preincubation also served to deplete any residual 02. Two types of incubation vessels were used; in the first procedure, 50 g of soil and 40 ml of water were added to 125-ml Erlenmeyer flasks and prepared in a manner identical to that described for the N03-IN02- experiments. In 37 Table 6. Characterization of soils. Organic Mineralizable matter carbona Series Texture Classification pH (%) (ugC/g) Brookston Loam Typic argiaquoll 7.0 3.2 152 Spinks Loamy Psammentic sand hapludalf 6.4 1.5 126 Miami Sandy Typic hapludalf loam 6.6 2.7 166 a Mineralizable C was determined by incubation according to the method of Burford and Bremner (2). 38 the second (and later used) approach, 10 g of soil and 10 ml of water were added to 27-ml Hungate tubes which were capped with Hungate stappers and aluminum seals. These tubes were then evacuated and filled with helium three times while agitating the tubes with a vortex mixer. The reasons for changing the conditions of incubation will be discussed in the Results section. Generation and preparation of 13N 13 N-labeled NO3- and N02- were produced at the cyclotron by the procedure described in the Appendix. The 13N species produced were 13 predominantly N03- (75-90%) with smaller quantities of 13N0 - and 2 13NH4+ also present in solution. The 13NH4+ was removed by the pro- cedure described in Chapter I. The 13N substrate was dissolved in water containing the desired amounts of nonlabeled (carrier) KNO or KNO . 3 2 13 . 13 - 13 The N substrate consisted of about 85—95% N03 and 5-15% N02. Since NO — is the first product in the reduction of N0 -, I assumed that 2 the 13N-NO3-INOZ- mixture was suitable for the experiments performed. Experimental procedure For experiments on the effect of N03- or N02- concentration, the continuous gas stripping system was used (see Appendix, Figure 18). The flask containing the soil slurry was placed on a magnetic stirrer and capped with a rubber stopper which contained a sparging tube and an outlet tube. The soil slurry was continuously stirred and sparged with helium at a rate of about 100 ml/min. The effluent gas passed through a differential trapping system to separate and quantify [13N]-N20 and [13N]-N2. For the higher concentrations of N03- or N02- (2 to 20 ppm-N) about 1 mCi of 13N-substrate (activity at time of addition) was present with the nonlabeled carrier in the 2-ml addition to each flask. For 39 lower N03- or NOZ— concentrations (0 and 0.5 ppm—N) smaller quantities of isotOpe (about 0.1 mCi) were used to avoid saturating the detectors. The concentrations of NO3_ and N02- employed are expressed as ppm-N on a moist soil basis (ug N/g soil). An activity of 0.1 mCi indicates the 13 presence of about 68 fg NOB-IIBNO “N, so that the 0 ppm addition 2 actually contained this amount of substrate plus any traces of N03- or NOZ- that occurred in the twice distilled, deionized water used. I assumed that trace quantities of N03- and N02- were also present in the soil slurries since it was not likely that the preincubation could have 13 reduced the concentration of these ions to the level of N added. Subsamples of the isotope solution (100 pl) were counted in a NaI(Tl) well counter to determine the total amount of 13N added to each flask. The half-life corrections required for counting the water sample, which also contained 18F, are detailed in the Appendix. Data collection began at the time of substrate addition and con- 3 was depleted. The data for [13Nl-N20 and [13NJ-N2 accumulation in the tinued (using 1 min summations) for about 1 hour or until the N03-INO traps were taken using a PDP 11/45 computer which performed on-line corrections for detector efficiencies, background, and isotope half— life. The corrected data was continuously displayed on a monitor and at the end of an experiment hard copy output of both corrected and uncor- rected data was obtained. The data collection procedure is described in greater detail in the Appendix. To study the effects of 02, varying quantities of 02 were injected into three or four of the soil slurries, which were then vigorously agitated to allow equilibration of the added 0 2. 13N substrate was injected (1 ml for flasks; 0.5 ml for Hungate tubes). About 1 min later, the 40 When noted, 0.1 ppm N03--N carrier was added with the 13N substrate. After varying times of incubation (2-20 min) on a rotary shaker, a l-ml (0.5-ml for Hungate tubes) sample was taken from the headspace of each container using a 1-ml syringe with gas-tight valve. The relative amounts of [13Nl-N20 and [13Nl-N2 in the samples were determined using the gas chromatograph-prOportional counter. Data were collected and stored using a Sigma-7 computer which was then used for off-line back- ground and half-life corrections and peak integrations. The micro- thermistor detector also enabled mass analysis of the gases (02, N2, N20, C02) when Sufficient quantity of gas was present. RESULTS Nitrite effect 13N-labeled gas production from the Spinks soil in the presence of varying amounts of added N02 carrier is shown in Figures 5, 6, 7 and 8. In Figure 5, no N02 carrier was added and N2 was by far the predominant product of denitrification. It is probable that denitrification of the very small quantity of labeled and residual, nonlabeled substrate was complete within a few minutes. The continued accumulation of [13N]-N2 until about 24 min, is probably an indication of the time required to completely strip the [13N]—N2 product from the aqueous phase and transfer to the Molecular Sieve trap. In the presence of 0.5 ppm N02--N (Fig. 6), N2 was again the predominant product, but a significant quantity of N20 was produced. The accumulation of labeled gas leveled out after about 40 min, indicat- ing that the denitrification of the added substrate was complete. Hence the actual concentration of added NOZ- decreased during the incubation "N COUNTS {10' DPM] 41 SPINKS + Oppm NO,“ [N] 3 d 13 [ NJ’Nzo 3“ :: u .. b n w :7 4:. :7 :7 x :7 :7 : x J 0 1 L 1 1 1 1 L 1 1 1 10 16 21 26 31 TIME AFTER "N ADDITION [MIN] Figure 5. 13N-gas production by the Spinks soil with the addition of 0 ppm N02 -N carrier. 42 ‘10 1 I T I I I I I I I SPINKS SOIL + U.Sppm N02 [N] (”NJ-N2 .. B 52 30 - >< 33 . 0. Q. U) 20 — '— 2 I3 8 [ NJ'NQO - L) = :4 ;:7:: A: : = -% ,5: 10 - ?—"—T—‘—i—’—T_'—Y . . 1 I. 1 T 10 16 29 32 90 98 TIME AFTER "N ADDITION [MIN] Figure 6. 13N-gas production by the Spinks soil with the addition of 0.5 ppm N02--N carrier. 43 from 0.5 ppm at the time of addition to about 0 ppm at the time of completion. It can be seen that in the early phase of the incubation (0-20 min) N O was actually the predominant product, but the accumula- 2 tion of N20 ceased at about 26 min while the N2 continued to accumulate, becoming the predominant product before its production stopped at about 40 min. 2 -N were added with the labeled substrate, N20 production exceeded N2 production. The addition When 2 (not shown) 4 ppm (Figure 7) N0 of 8 ppm N02--N resulted in even less N2 production relative to N20 (Figure 8). It is apparent from the data in Figure 5, that in the absence of added carrier, valid rates of N2 and N20 production were not established before the substrate was depleted. Hence, to compare the production of N2 and N20, I have compared the quantity of [13N]-N2 and [13Nl-N20 produced by a given time after the addition of substrate (40 min). At the higher concentrations of N02_ and N03“ (2 ppm N and above), the use of rates of gas production at 40 min would give similar results to those obtained by using the quantities of labeled gas produced at 40 min. A summary of the effect of N02- concentration on the gaseous products of denitrification in two soils is presented in Table 7. In both the Brookston and Spinks soils, N2 was the dominant product in the absence of added carrier N02- or in the presence of very low (§_0.5 ppm) N02-. At concentrations of NO - of 2.0 ppm-N or higher, N O became the 2 2 ’predominant product. At N02- concentrations of 0.5 ppm-N and above, linear rates of denitrification were established. In the Spinks soil this was about 1 ug N-g-l-hr-1 and did not vary significantly for the range of NO2 "N COUNTS [10' DPM] 44 Gobl I l I T— V I I I SPINKS SOIL - + prm NO,’ [N] (I 45- . (”NJ-N20 30- 15- _ (”NJ'Nz I: . - ._ ...-. = ” ”‘ " 1 L 1 1 1 1 29 36 98 60 TIME AFTER l“N ADDITION [MIN] Figure 7. 13N-gas production by the Spinks soil with the addition of 4.0 ppm NO2 -N carrier. "N COUNTS (10' DPM) 45 IBPT T 1 T I r [ T r _. SPINKS SOIL - + 8ppm NO,’ [N] 4 12- .1 al- (”NJ’Nzo _ 4L. _ [IJNl-Ng _ d o -- _-__-- l 24 36 98 60 TIME AFTER "N ADDITION [MIN] Figure 8. 13N—gas production by the Spinks soil with the addition of 8.0 ppm NO --N carrier. 2 46 Table 7. Effect of N0 - concentration on N O/N . 2 2 2 _ Denitrification 13[N]-N20 NO2 -N . rate N20 N2 ‘I§-*--- (ppm)a (Ug N°g soil—l-hour-l)b -(% of total 13N gasc)- [Nl-NZ Eagle -0 - 11.5 88.5 0.13 0.5 1.18 , 4 26.0 74.0 0.35 2.0 1.29 54.9 45.1 1.2 4.0 1.08 88.7 11.3 ‘ 7.8 8.0 0.98 94.8 ~ 5.2 18.1 Brookston 0 - 1.4‘ 98.6 0.01 0.5 - 14.1 " 85.9 0.16 2.0 p - 30.6 69.4 0.44 6.7 - ~ 59.2 ‘ 40.8 1.4 20.0 - 84.7 15.3 5.5 a I ug NO --N/g field moist soil. The water content of the field moist soil was 4.3% - Spinks and 15.3% - Brookston. Calculated as: Adpm 13H gas'hr-1°tota1 dpm added-l-ppm-N added; for the 20-40 min time interval in the 2,4 and 8 ppm additions; for the 20-30 min interval in the-0.5 ppm addition. Total dpms added not available for Brookston soil. c Taken from total disintegrations of each gas after 40 min of incubation. 47 concentrations used. Hence there was no independent effect of the N02 concentration on the denitrification rate. This also suggests that the - was below the NO I concentrations used. apparent Km for N02 2 Nitrate effect A summary of the results of studies on the effect of NO3 concen- tration on the ratio of NZO/N2 production is shown in Table 8. Increasing the concentration of carrier N03- added, resulted in increased N20 2. But the effect of the increased N03- con- 2 O the highest concentration of N03- used (20 ppm-N), N2 remained the production relative to N centration was not as large as was the effect of added NO Even at dominant product of denitrification. 3 was added to the soils (Figure 9). During the early period (10-15 min) A distinct pattern of gas production was observed when 20 ppm N0 of incubation a high rate of [13Nl-N2 production was apparent. This then decreased to a slower linear rate which continued until the experi- ment was terminated. This same pattern occurred in three 20 ppm NO3 -N 3 -N experiment with the Spinks soil. A similar pattern was also observed experiments performed with the Brookston soil and in the one 20 ppm NO in an 8 ppm NO3 -N incubation with the Brookston soil but was not apparent in the 2 ppm NO3 -N incubations (Figure 10). This pattern was also not observed in the 20 ppm NO --N addition to the Brookston soil. 2 During the period of rate decline in [13Nl-N2 production a slight increase in the rate of [13N]-N20 production did occur (Figure 9). But the increase in rate of N20 production was not as large as was the decline in rate of N2 production. In Table 8 it can be seen that the overall denitrification rate did respond to the increase in N03 concentration. In both soils, an 48 Table 8. Effect of N03- concentration on N20/N2. N03_-N Denit::::cation N20 N2 13[N]-N20 (ppm)a (pg N-g soil-l'hour-l)b -(% of total 13N gasc)- l3[N]-—N2 sale 0 - 4.8 95.2 0.05 0.5 0.54 6.1 93.9 0.07 2.0 0.73 10.2 89.8 0.11 20.0 1.15 14.6 85.4 0.17 Brookston 0 - 1.3 98.7 0.01 0.5 0.70 6.1 93.9 0.06 2.0 1.14 10.9 89.1 0.12 20.0 1.68 19.0 81.0 0.23 3 pg N0 --N/g field moist soil. The water content of the field moist soil was 4.3% - Spinks and 15.3% - Brookston. Calculated as: Adpm 13N gas'hour-1:total dpm added-l-ppm-N added; for the 30-40 min time interval on all except the Brookston 0.5 ppm, for which the 25-35 min interval was used. Taken from total disintegrations of each gas after 40 min of incuba- tion. "N COUNTS [10° DPMJ 49 H— r 1 T j T 1 T I T BROOKSTON SOIL . ° F + 20ppm N03“ [N] , " - 31- 3 _. . [I NJ’Nz 2L _ L . 1- [IIPQJ’PN2EJ q E T '10 L 210 1 310 l 910 l 510 I1 TIME AFTER "N ADDITION [MIN] Figure 9. l3N-gas production by the Brookston soil with the addition of 20 ppm NO --N carrier. 3 18_ I I I I r I I T --i BROOKSTON SOIL _ + 2ppm NO.” [N] 4 E12»- , 7 CL C] 7&2 L T C...’ [‘3N1—N2 00 3 A (~2- F 8 L) - '1 Z '3 H- -+ ("NJ-N.O o - 1...... A 1 L L L 1 I 0 13 28 39 52 TIME AFTER "N ADDITION [MIN] Figure 10. N-gas production by the Brookston soil with the addition of 2.0 ppm NO3 —N carrier. 51 increase in rate of gas production was observed between 0.5, 2.0 and 20 ppm-N. The maximum rate of gas production in the Spinks soil (1.15 ug N-g-l'hour-l) was about the same as that reported in Table 7. This suggests that the rate of denitrification increased with increasing NO3 concentrations until the rate of reduction of N02- became rate limiting. Oxygen effect As described in the Materials and Methods section, two types of soil slurry containers were employed for these experiments. In the first procedure used, 50 g of soil and 50 m1 of water contained in 125 ml Erlenmeyer flasks, were incubated at 250 rpm on a rotary shaker. A sample of the data obtained using this procedure, is shown in Figure 11. The denitrification activity decreased tremendously when 02 was intro- duced (see also Table 9). But of greater interest, is that the percentage of product accumulating as [13Nl-N 0 increased with the increasing 2 additions of O . 2 The data obtained using this incubation procedure were inconsistent and difficult to reproduce. The problems were caused by two factors. First, the soil slurry volume was large with respect to the gas-liquid interface area and the slurry was not thoroughly mixed. Secondly, the headspace volume sampled for analysis was large (around 70 ml) while the maximum size of sample that could be analyzed with the GC-PC system was 2 m1. When 02 was added to the incubation, the denitrification activity was so low that frequently valid quantfication of the [13Nl-N2 produced could not be achieved. An improved incubation procedure was adopted in which 10 g of soil and 10 ml of water were incubated in 27-ml Hungate tubes. The tubes were then incubated in a horizontal position with vigorous agitation on 52 EFFECT OF 02 ON DENITRIFICATION ACTIVITY AND PRODUCTS IN BROOKSTON SOIL q IOO .60? 4. "é :- 0/0 N20 X | 80 g, o : e 2‘“ 40 o 2 T 60 g a) Q s 8 22 'u ‘ 40 g; S 20 T : °‘° L . 20"; - <1 \\\ ~Total Denitrification '5 _____________ _O +— 1 1 1 1 1 O 0 0.04 0. l2 0.20 02 ADDED (ATM) Figure 11. Influence of 02 on denitrification activity and products in a Brookston soil. 53 Table 9. Effect of O 03 denitrification activity and products in a Brookston soil . 02 added [13N]-N20 [13Nl-N2 Total 13N-gas % of 13N gas (10"2 atm) (102 disintegrations) as N20 0 560 (3.5) 1340 (5.9) 1899 29.5 1.6 176 (2.5) 198 (3.0) 374 47.1 16.3 65 (2.0) 47 (2.0) 111 58.0 a 50 g soil + 50 ml H 0 in a 125 ml Erlenmeyer flask. 2 54 a rotary shaker. This maximized the gas-liquid interface and minimized the slurry depth, thus improving diffusion of 02 into the soil slurry and of product gas out. The volume of the headspace was about 12 ml, which allowed analysis of a larger portion of the total gas in the headspace. Data obtained using this procedure with a Miami soil are shown in Figures 12 and 13. In Figure 12 (and Table 10), it can be seen that the percentage of product occurring as [13Nl-N20 increased with increasing 02. In the 10 min sample, in the absence of 02, [13Nl-N20 constituted 14% of the total 13N-gas; while in the presence of 0.05 atm 02, 73% of the 13H product was as [13N]-N20. It is again apparent in Figure 13 (and Table 10) that introduction of even a small quantity of 02 (0.017 atm) caused a significant decrease (l7—fold) in denitrification activity. One can compare the results obtained in Figures 13 and 11 and note that in the "improved" incubation procedure (Fig. 13), concentrations of 02 greater than 0.05 atm could not be tested because the denitrification activity was too low for accurate analysis; while in the original incubation procedure (Figure 11), denitrification activity could be measured in the presence of 0.20 atm 02. It is apparent that rapid and complete equilibration of 02 with the entire soil slurry significantly affected the results. It should be understood that 02 was being consumed during the incubations. Hence the percentage of product as N 0 in the presence of, 2 for example, 0.05 atm 02 is not predictive of an absolute ratio of N20/N2 to be found under these 02 conditions. But the data do indicate the pattern of product composition to be expected as the availability of 02 increases. 55 EFFECT OF 02 ON N20 AS PRODUCT IN MIAMI SOIL 80C A % 0F '3N GAS AS N20 .4> (II 0 O [\D C) 0 0.02 0.04 0.06 02 ADDED (ATM) Figure 12. Influence of 02 on the product composition of denitrification in a Miami soil. 56 EFFECT OF 02 ON DENITRIFICATION ACTIVITY IN MIAMI SOIL 300 - B 200 |00 TOTAL '°N GAS PRODUCED (DPS) x IO3 O 0.02 0.04 0.06 02 ADDED (ATM) Figure 13. Influence of 02 on denitrification activity in a Miami soil. 57 .onsu oumwoom HBINN m :8 O N: as S + :68 m S m H.8m ow Am.Hv mm Am.Hv he o.m 8.0m mma AN.NV mm Am.Nv om m.m «.mm Nmmm Aa.oav mnwm Am.NHv mHHN N.H 0N m.NN mm A¢.ov Ha an.ov wN o.m «.mo 8N An.ov mN AN.HV Hm m.m N.mm ooN Aw.av wNH AH.NV Noa N.H m.mH oqu A¢.HHV mmoq Aa.mv ans 0 OH 0N2 mm Awaowumuwmucfimflo NOHVIIIIIIIIII Aaum NICHV AGHEV N N N Canaan mo mafia w o m w I I o o mm ZMH mo N o ZNHHmuOH z HZNHH o z HZNHH o oo 0 .H808 Hana: m a“ muoooouo pom >u8>fluoo oceanogmwuuwaoo no coaumuooooooo No mo mocmoamoH .oa Danae 58 DISCUSSION The data presented in Chapter I indicate that N20 is generally a free intermediate, able to diffuse away from the site of active denitrifi- cation. Thus any factor which increases the rate of NO3-/NO2 reduction to N20, without comparably increasing the rate of N 0 reduction, could 2 result in loss of N20 from the reductive process. Similarly, any factor which decreases the rate of N20 reduction, without decreasing the rate of N03-IN02- reduction, could yield the same result. The data reported in this Chapter show that as the concentration of, N02- increases, the prOportion of N20 occurring as the gaseous product of denitrification increased. This was not due to an increase in the rate of N20 production, for the total rates of N 0 + N production did 2 2 not vary significantly over the range of N02- concentrations used. The net result of an increase in N02- concentration must have been a decrease in the rate of N 0 reduction to N . It is possible that NO - directly 2 2 2 or indirectly inhibits N20 reductase. It is also possible that NO 2 diverts the reducing power available for N20 reduction to its own; however, the observation that the rate of N O + N2 production did not 2 increase with increasing NOZ- concentration argues against this explana- tion. I have also found that an increase in N03- concentration resulted in an increase in the N20/N2 ratio. In this case, the rate of gas formation did increase with increasing substrate (N03-) concentration. Several investigators have suggested that N03- is preferentially used over N20 as an electron acceptor (3, 5, 9, 11) but with no direct evidence that this was indeed the mechanism. Payne and Riley (10) have 59 reported that N03 does not inhibit N20 reductase in crude enzyme preparations from Pseudomonas perfectomarinus. This would be consistent with N03- acting as a competitive electron acceptor to N20 reduction. It may be impossible to determine the mode of action of N03- or N02- using soil systems. A possible approach to this question appears to be the use of bacterial strains devoid of N03- and/or N02 reductase activity. can- 3 O as the product of A number of early investigators reported that increasing NO centrations resulted in a higher proportion of N2 denitrification (3, 4, 9, 12). However, I have found that the influence 2 0 concentrations of N02- (0.5 ppm-N and below) have a profound influence of N03- is very small compared to the effect of N0 Even very low on N20 accumulation. One then must question whether N03-, per se, is an effector or whether it is actually the N02- produced from NO3- that is causing N20 accumulation. Recent work by Blackmer and Bremner (1) indicated that N03- concentrations as low as 10 ppm-N inhibit N20 reduction; my work indicates that the accumulation of even a small fraction of this N03- as N02- could cause the observed results. Accumula- tion of large N02- pools is not commonly observed in soils. However, the occurrence of very small N02- pools (near the detection limit of traditional analytical procedures) would be expected to influence N20 4 accumulation. COOper and Smith (4), who reported an increased prOportion of N20 with increasing N03 concentrations, also reported NO2 accumula- tion during the period of enhanced N 0 production. 2 It is possible that an accumulation of NO 2 results shown in Figure 9. The rate of N20 reduction to N2 may have been higher very early after the addition of 20 ppm N03-. A small pool caused the biphasic 60 of N02- may have accumulated and reduced the rate of N20 reduction to N This proposed accumulation of a NOZ- pool is consistent with the 2. data indicating that the reduction of N0 2 was the rate limiting step 3 . tion in the Spinks soil increased with increasing NO3 concentration during the reduction of 20 ppm NO That is, the rate of gas produc- 2 . It is well known that 02 strongly influences denitrification. The kinetic model proposed by Focht (6), based largely on Nommik's early until it reached the reported rate of gas production from N0 work (9) predicted that aeration status of soils should influence the NZO/N2 ratio. Work by Cady and Bartholomew (3) also indicated that 02 status influenced the relative production of N 0 and N . However, the 2 2 work by Nommik (9) and Cady and Bartholomew (3) employed long-term incubations (first sampling time at 2 days) in closed systems with large quantities of 02. In these extended incubations it was quite possible that the influence exerted by 02 was indirect. The presence of different 02 concentrations over a 2 to 99 day incubation could be expected to alter the microbial composition of the soil, influence the concentration of N03- available (if only through nitrification), and strongly influence the availability of carbon. My work indicates that the presence of 02 does strongly and directly influence the N20/N2 ratio of denitrification. The effect of 02 could result from a number of mechanisms including: 1) competition as an electron acceptor; 2) inhibition of N20 reductase; 3) a general overall slowdown of the denitrification process giving N20 more time to diffuse away from the active site; or a combination of these or other less direct influences. Because it is probable that much denitrification occurs in soils of mixed aeration status, 0 may well be 2 61 an important parameter influencing not only the quantity of denitrifica- tion occurring but the composition of the gas produced. I 1 71‘s.]! 10. 11. 12. LITERATURE CITED Blackmer, A. M. and J. M. Bremner. 1978. Inhibitory effect of nitrate on reduction of N 0 to N by soil microorganisms. Soil Biol. Biochem. 10:187-191? 2 Burford, J. R. and J. M. Bremner. 1975. Relationships between the denitrification capacities of soils and total, water-soluble and readily decomposable soil organic matter. Soil Biol. Biochem. 7:389—394. Cady, F. B. and W. V. Bartholomew. 1961. Influence of low p02 on denitrification process and products. Soil Sci. Soc. Am. Proc. 25:362-365. Cooper, A. S. and R. L. Smith. 1963. Sequence of products formed during denitrification in some diverse western soils. Soil Sci. Soc. Amer. Proc. 27:659-662. Delwiche, C. C. 1959. Production and utilization of nitrous oxide by Pseudomonas denitrificans. J. Bacteriol. 77:55—59. Focht, D. D. 1974. The effect of temperature, pH and aeration on the production of nitrous oxide and gaseous nitrogen - a zero-order kinetic model. Soil Sci. 118:173-179. Focht, D. D. and A. C. Chang. 1975. Nitrification and denitrifica- tion processes related to waste water treatment. Adv. in Appl. Microbiol. 19:153-186. Focht, D. D. and W. Verstraete. 1977. Biochemical ecology of nitrification and denitrification. Pages 135—214 in M. Alexander, ed. Advances in Microbial Ecology, vol 1. Plenum Press. Nommik, H. 1956. InvestigatiOns on denitrification in soil. Acta Agr. Scand., 6:195-228. Payne, W. J. and P. S. Riley. 1969. Suppression by nitrate of enzymatic reduction of nitric oxide. Proc. Soc. Exptl. Biol. and Med. 132:258-260. Payne, W. J. 1973. Reduction of nitrogenous oxides by microorganisms. Bacterial. Rev. 37:409-452. Wijler, J. and C. C. Delwiche. 1954. Investigations on the deni- trifying process in soil. Plant and Soil 5:155-169. 62 CHAPTER III TEMPORAL CHANGES IN COMPOSITION OF GASEOUS PRODUCTS OF DENITRIFICATION FOLLOWING ONSET OF ANAEROBIC CONDITIONS . . . . 13 During early studies on denitrification uSIng N, I noticed that the ratio of N20/N2 produced by soil appeared to change in response to the length of the time that the soil had been anaerobic. The predominant product for the first 1 to 2 hours was N ; thereafter the proportion of 2 N20 increased with N20 often becoming the dominant product (12). It seems quite reasonable that changes in the soil environment and/or changes in members of the microbial community or their physiology would occur in response to the shift to anaerobiosis and that these changes could alter the ratio of NZO/NZ' The environmental parameters which could change with time after the onset of anaerobiosis are NO3 concen- tration, N02- concentration, carbon availability and redox potential. Changes in the microbial community could include relative shifts in density of species, increased numbers of denitrifiers, and increased denitrifying activity due to derepression and synthesis of denitrifying enzymes. The effect of length of anaerobiosis on the products of denitrifica- tion was examined using the acetylene inhibition technique. The inhibition by acetylene of N20 reductase in pure cultures was reported by Yoshinari and Knowles (15) and by Balderston gt 31. (1). In the presence of sufficient acetylene and NO -, N O is the sole product of denitrifica- 2 tion (1, 12, 15, 16). Analysis is greatly simplified because N O, 2 unlike N2, is a minor atmospheric constituent (approximately 300 ppb) and can be assayed by sensitive gas chromatographic detectors. The 63 64 assumption is that the quantity of N20 produced in the presence of acetylene is a direct measure of the total gaseous N that would be produced in the absence of the inhibitor. Using 13N techniques this assumption has been confirmed by Smith e£_el. (12). The successful application of the acetylene inhibition method to soil denitrification studies has been reported by several groups (5, 12, 16). To determine the composition of the gaseous products of denitrification one can determine the quantity of N20 produced in the absence and in the presence of acetylene. The assumption is that the ratio of the quantity of N O 2 produced in the absence of acetylene to the quantity of N 0 produced in 2 the presence of acetylene, accurately reflects the NZO/(NZO + N2) ratio that would occur in the absence of the inhibitor. This assumption has also been confirmed using 13N methods (12). By using the acetylene inhibition method I have recorded the changes that occur in the N20/(N20 + N2) ratio with time after the onset of anaerobic conditions in soils and in a bacterial culture and have attempted to determine the factors responsible for these changes. MATERIALS AND METHODS Soils used and preparation The soils used in this study were a Brookston loam, a Spinks loamy sand and a Conover loam. The characteristics of the Brookston and 'Spinks soils were reported in Table 6 of Chapter II. The Conover soil, classified as a Udallic ochraqualf, had a pH of 6.8 and contained 3.1% organic carbon. After collection, the moist soils were passed through a 5 mm sieve to remove rocks and debris and stored in sealed plastic bags at 2° C until used. 65 For all soils experiments (exCept those requiring removal of indigenous N03-), 50 g (fresh weight) of soil and 50 ml of H20, con- taining the desired quantity of KNOB, were added to 125-ml Erlenmeyer flasks which were sealed with rubber stappers pierced by a glass tube capped with a serum stapper. The soil slurries were made anaerobic by evacuating (to 30 inches Hg) for 3 min and filling with He, using a multiple gassing manifold. This procedure was repeated three times and required a total of 10 min. The time at the end of the third evacuation was recorded as the zero-time for the anaerobic incubation. Seven milliliters of acetylene (99.6%, Matheson Gas, Joliet, 111.) was added to flasks to be used for assay of denitrification rate. The flasks were incubated on a rotary shaker at 250 rpm at room temperature. All treat- ments were replicated three times and the data reported are means of the three replicates. Chloramphenicol (Sigma, St. Louis, Mo.) was added where indicated during an incubation by removal of the rubber stapper and addition of 0.25 g of the antibiotic. The flasks were then refilled with He. The solubility of chloramphenicol is 2.5 mg/ml; the concentration used in the soil experiments was about twice its solubility. I assumed that a portion of the antibiotic added would be bound to soil surfaces. I hoped to supply enough excess chloramphenicol to maintain a high con- centration in solution. When the addition of glucose was required during an incubation, 1 m1 of a stock solution under He was injected into the flasks to give a final concentration of about 0.1%. When it was necessary to remove indigenous soil N03- and N02- before the beginning of an experiment, 50- 66 g (fresh weight) samples of soil were extracted with 75 m1 of H20 three times. The procedure required approximately 1 hour. The concentrations of N03- are expressed as ug—N/g fresh weight soil. Gas chromatqgraphic analyses A Perkin-Elmer model 900 gas chromatograph (Norwalk, Conn.) with a Hot Wire detector was used for most of the gas analyses. The lower limit of detection for N20 was about 10 ppm (v/v). A Parapak Q column (3 mm x 1.8 m) at ambient temperatures, with a He-carrier gas flow of 15 m1/min was used to separate N20, C02, acetylene and air (02 and N not 2 resolved). Peak areas were determined with a Spectra Physics, System I computing integrator (Chicago, 111.). Standard curves were determined for every experiment run, using prepared N20 standards. Samples of the headspace gas (0.5 ml) were periodically removed with a 1-cc syringe equipped with a Pressure-lock valve. In all experi- ments, the concentration of N20 in solution was calculated from the measured headspace concentration, and the quantities of N 0 corrected 2 accordingly. We had previously verified that the published values of the Bunsen absorption coefficient (0.66) approximated the N O solubility 2 in the soil slurries used (12). The rate of denitrification was determined by measuring the rate of N20 production in the presence of 0.1 atm of acetylene and the net rate of N20 accumulation was determined in the absence of acetylene. The ratio of these two rates of N20 production (absence of acetylene/presence of acetylene) should represent the proportion of the total gaseous products of denitrification appearing as N20. 67 Bacterial culture experiments Flavobacterium sp. #175 was used in the experiments reported here. The cells were grown aerobically in 500 m1 of 3% Tryptic Soy Broth (Difco, Detroit, MI), in a 2-1 Erlenmeyer flask on a rotary shaker at 300 rpm, at 28° C for 12 hours. The cells were harvested and washed as previously reported in Chapter I and resuspended in 0.02 M phosphate buffer, pH 7.2. Five milliliters of this cell suspension were added to 27-m1 serum vials. One milliliter of a solution containing 1% glucose, 60 mM KNO3 (when desired), and 1.2 mg of chloramphenicol (when required was added); all treatments were Performed in duplicate. The vials were capped with Hungate septa and sealed with aluminum crimp seals. Each vial was then evacuated and filled three times with He. One milliliter of acetylene was added to the vials containing NO3_. To some of the vials without NO3-, 0.5 ml of N20 was added, but to others, no electron acceptor was added. The vials were then incubated at room temperature on a rotary shaker at 250 rpm. At the desired time intervals, 0.2—m1 samples were taken from the headspace gas and analyzed by gas chromatography. The data analysis and gas solubility corrections were essentially the same as those used for the soil slurries. RESULTS An example of the data on rates of denitrification and N20 produc- tion by a soil slurry at various times after the imposition of anaerobic conditions is shown in Figures 14 and 15. Each data point is the mean of three replicates. The overall rate of denitrification (as determined in the presence of acetylene) increased during the first 4 hours until 68 .NmNo mo mocomom mam mocomopo OSO ow sawomoDOCN mo mommca zaumm wowuao HHom coomxooum m :w ONz mo coNuoooon .OH muowam :52: mi; 8 cm 8. m. o c o 1 — — — _ A 8 22.8382: \\ 882 52. am \.\ I _ an x u. w x \ O a .. . ~z~o+ \ s x\ ON x 20:48:58..wa \rc «zuo + x ON 69 .NmNo mo oucmmnm pom mocomoua mcu aw Hwom COOmxooum a Ca mafia :uwB oNz mo cowuoapouo omscfiucoo .mH ouowwm :52: NE... mm mm m¢ mm cm on 1 FOIL fix a _ q .\ I.¢w x\ z x IO\O\O\QI \ O . .6. 8 v.\.. ) a. .1 8+ 9A 18:" N N H 1 0+ 0 ql x q. X\\X\\. unavN"l\ \ \x {NINUIT 70 it reached a relatively constant rate after about 5 hours. The rate of N 0 production also increased during the early period of incubation, 2 reaching a relatively constant rate after 4 to 5 hours, which persisted through the end of the first 12 hour period. To avoid any long term effects of the CZHZ treatment, another set of 6 flasks which had been anaerobic for 16 hours, was reevacuated and filled with He, and CZHZ was added to three. The gas production in these flasks was then followed from 18 hours to 24 hours. Another set of flasks was started at 26 hours. It is apparent from Figures 14 and 15, that the rate of deni- trification continued to exceed the net rate of N20 production during the 18- to 37-hour period. It can also be seen that the net rate of N20 production declined between 28 to 30 hours. In another set of flasks to which acetylene was first added at 48 hours, it can be seen that while denitrification continued during the 48 to 52 hour period, N20 was no longer accumulating. The rates of N20 production and denitrification shown in Figures 14 and 15 were quantified using linear regressions and are reported in Table 11. To determine if long-term exposure to CZHZ influenced the denitrification rate, C2H2 was added to an alternate set of three flasks which had been anaerobic for 8 hours. The rate of denitrification determined for the 8 to 12 hour period was 54.7 nmoles-g-l'hour-l, very close to the value derived from the flasks previously exposed to C2H2 for 8 hours (51.2 nmoles‘g-l-hour-l). Both N20 production and denitrification reached maximum linear rates during the 7- to 12-hour period. The rate of denitrification then decreased until about 26 hours; from 26 to 53 hours a relatively constant rate of denitrification (24-25 nmoles'g-l'hour_l) was maintained. 71 Table 11. Rates of N 0 production and denitrification following the onset of anaerobic conditions in a Brookston soila. Net rate of Rate of Fraportion of Time period N20 production denitrifie:tion total proguct (hours) ---(nmoles gas-g soil -hour )-- as N20 3-5 15.7 (0.99)C 34.1 (0.99) 0.46 7-12 24.4 (0.99) 51.2 (0.99) 0.48 18-23 19.7 (0.97) 41.2 (0.98) 0.48 26—29 11.5 (0.60) 24.2 (1.00) 0.48 33-37 5.0 (0.89) 25.2 (0.97) 0.20 48-53 -l.l (0.21) 24.2 (0.98) N20 consumption a At zero-time the Brookston slurry contained 107 ppm NO --N. (7 ppm already present; 100 ppm added). incubation, 39 ppm NO b Fraportion of total product as N % (rate of denitrification). c Coefficient of determination (r2). 3 -N remained. O = (net rate of N 3 After the 53 hour anaerobic 20 production) 72 During the period of 12 to 53 hours the net rate of N20 production continuously declined until at 48 hours a negative value was determined. This negative value for the net rate of N20 production indicates that N20 reduction activity exceeded N20 production activity. Under these conditions the soil would serve as a net sink for N20. ’During the first 29 hours, N20 comprised a relatively constant proportion of the total denitrification product (about 48%). During the 33 to 37 hour period, this had declined to 20%, and by 48 hours, N20 was being consumed. This net consumption of N20 after 48 hours was not due to depletion of N03- because 39 ppm N03_-N remained at the end of the experiment (53 hours). The results from a similar experiment with a Miami soil are shown in Table 12. In this soil the maximum net rate of N20 production occurred between 23 to 26 hours. Between 32 to 36 hours, no increase in N20 was observed and by 47 hours N20 was being consumed. Before 26 hours, N20 was the dominant product of denitrification; after 32 hours, no net N20 production was observed. As similarly reported for the Brookston sail, a dramatic decrease in prOportion of gaseous product as N20 occurred between 26 and 32 hours after the onset of anaerobic conditions. It seemed plausible that the decrease in net production of N20 may have been due to an increase in N O-reducing activity. To determine 2 whether ge_novo synthesis of an enzyme was responsible for the observed phenomenon, an inhibitor of protein synthesis (chloramphenicol) was added to the soil prior to the decrease in net N 0 production. The 2 results of this experiment on a Conover soil are shown in Table 13. In the absence of chloramphenicol, a pattern of net N20 production similar to that reported for the Miami and Brookston soils, was observed. The 73 Table 12. Rates of N 0 production and denitrification folloging the onset of anaerobic conditions in a Miami soil . Net rate of Rate of Proportion of Time period N20 production denitrifiiation total product (hours) ----(nmoles gas-g soil -hour )---- as N20 3-5 5.0 (0.94)b 9.1 (0.96) 0.55 9-12 14.1 (0.97) 15.2 (0.92) 0.93 23-26 62.9 (0.92) 53.2 (0.89) 1.18 32-36 0.0 (0.00) 36.2 (0.93) 0.00 47—51 -26.0 (0.99) 62.9 (0.97) N20 consumption a At zero-time the Miami slurry contained 100 ppm NO3--N. After the 51 hours anaerobic incubation, 59 ppm NO3 -N remained. b Coefficient of determination (r2). 74 Table 13. Rates of N 0 production and denitrification in the 2 presence and absence of chloramphenicol in a Conover soila. Net rate of Rate of Proportion of Time period N20 production denitrification total product (hours) ----(nmoles gas-g soil -hour )----- as N20 1-2 9.0 (0.99) 9.6 (0.99) 0.93 3-4 12.2 (1.00) 13.5 (1.00) 0.91 5-10 21.2 (0.94) 25.5 (0.99) 0.83 22—25 4.5 (0.99) 17.4 (1.00) 0.26 27-30 -5.1 (0.21) 7.7 (0.99) N20 consumption 32—36 0.5 (0.01) 12.8 (0.78) 0.04 45-49 4.7 (0.36) 47.4 (0.92) 0.10 + chloramphenicol at 22 hoursb 26-30 7.9 (0.99) 8.5 (0.92) 0.92 33-36 7.7 (0.88) 9.1 (0.98) 0.84 46-49 11.1 (0.94) 16.8 (0.80) 0.66 a At zero-time the Conover slurry contained 137 ppm NO --N. After 3 49 hours anaerobic incubation 108 ppm NO --N remained. 3 b 0.25 g of chloramphenicol was added to each flask. 75 net rate of N 0 production exhibited a sharp decrease between 10 and 22 2 hours, as did the proportion of gaseous product as N O. In the soils to 2 which chloramphenicol was added at 22 hours, the net rate of N O produc- 2 tion did not similarly decrease; in fact, N O remained the predominant 2 product of denitrification through the end of the assay at 49 hours. Chloramphenicol was also added to the Brookston soil prior to the decrease in net N20 production. The rates of gas production observed during the 30 to 47 hour period in the presence and absence of chloramphen- icol are reported in Table 14. There was no significant difference in the rates of denitrification found in the presence and absence of the inhibitor, but in the presence of chloramphenicol a much higher net rate of N20 production was determined. A similar effect of chloramphenicol was also observed in the Miami soil (data not shown). In the presence of the inhibitor of protein synthesis, N20 remained the dominant product of denitrification. It appeared as though the decrease in the net rate in N20 production resulted from an increase in N20 reducing activity and that this increase in N20 reduction required de novo synthesis of protein. To determine if the presence of N03 or N20 was required to produce the characteristic decrease in net rate of N20 production, the following experiment was performed. The Brookston soil with indigenous NO3 + N02--N removed was incubated with NO3-, N20 or no added electron acceptor. NO3- was then added to the non-N03- amended flasks at 22 hours, so that the two activities could be assayed. The results are reported in Table 15. In the presence of NO3 , the sharp decline in the net rate of N20 production occurred between 12 and 23 hours. A similar decrease was observed in the soils incubated with N20 for 22 hours. However, in the soils incubated anaerobically, in the absence of NO3 and N20, the net 76 .OGONquooo ownouomam mo oomoo may Haumm whoa; HN um xmmaw some cu vooom was NOONconmfimuoano mo w mN.o m 5H.o Aoo.Hv w.H8 Amw.ov 0.5 melee I om.o Amm.ov H.Hm Amm.ov o.mH «NIom I 05.0 flaw.ov n.08 Aww.ov N.wN Nchq + mn.o Aom.ov N.mq Ama.ov H.Nm SmIom + oNz mm IIIAaIuoon.HIHHow m.mmw ooaoaovllll Amumonv maooanoneamuoano N oofiuo 8885 uoaoouo Hauou cowumONMNuufioOC coeuospoua o 2 mo oowuuoooum mo Doom mo mums uoz mo ooaomom pom oooomouo Ono :8 coauOONMNHuNGOC pom Gowoosooua o .Hfiom caumxooum m :8 Hoowaonmamuoano NZ 86 88888 .SH 88888 77 .mmmmm now whoa: NN um compo 883 ZIImoz and 008 n .xmmmm you women was ZIImoz Eon 00H .muson NN u< .OBHU anon um compo 0803 oNz mo moaofi: NH 8 mw.o Awm.0v N.Nm Ama.ov m.Nm Nelnm mm.o Amm.ov m.w8 Aoo.HV m.Nn oNINN oa8o anon on omoom Hoodoooo couuooao oz 86888688866 o«z 188.88 8.88 x88.88 8.8- om-«8 86888588866 o«z 188.88 8.8« a8«.ov «.8- «8-88 HN.o Acm.oV 8.88 Aow.ov o.m8 oNImN moafiu anon 8 Canon 0 2 86888888866 o«z A8«.ov 8.«8 Am8.88 8.««- 88-88 86888888866 o«z A«8.88 «.88 fi8«.88 8.8- 88-88 wo.o Acm.ov 8.8m Anm.ov N.8 mNImN om.o Aoo.dv H.Nw Amm.ov m.w8 NHIm mm.o Aoo.8v m.HN Awm.oV H.mN «IN m~.o Ama.ov n.8m Amm.ov 8.8 N.Hln.o CMHu anon on z- 82-888 888 oNz mo IIIII AHIuaon. IHHOO m.mow mafioaavIIIII Amuooav muoOoooom :OHuOOHO oooooua Hmoou GONuMO8MHuu8ooo doauoavouo oNz powuoo Oe8H mo moumum mo ao8uuoaoum mo oumm «a moon uoz .HHOO naumxooum o :8 .o z 8883 8:8 .-8oz 8888883 . moz nufis pawuonoocfi O8nouomcm uoumo nowuoONMNuufioop one dowuoooouo o 2 mo moumm .nH OHAOH 78 rate of N20 production remained high through 26 hours and N20 was the predominant product of denitrification from 22 to 42 hours. Hence the presence of N03 or N20 during the first 22 hours of incubation was required to produce the observed decrease in net rate of N20 production. It is probable that N20 or N03- induced or enhanced synthesis of an enzyme involved in N 0 reduction. 2 It seemed very likely that the readily available carbon present in the soil slurries decreased with time of anaerobic incubation. To determine if carbon depletion influenced the composition of the gaseous products of denitrification, glucose was added to the Brookston soil at several times during the anaerobic incubation. As can be seen in Table 16, in the absence of added carbon the characteristic pattern of N20 evolution was observed. The addition of glucose immediately prior to the period of assay, produced several effects: First, the overall rate of denitrification increased (about 3—fold) in the presence of glucose, indicating that the process was carbon limited. Second, the prOportion of product as N 0 did not significantly decrease after 23 hours. The 2 addition of glucose caused increased production of N20 relative to N2. The data thus far presented indicate that after a period of anaerobic incubation in the presence of N03 or N20, the rate of N2 increases, relative to the rate of N20 production. If the factor 0 reduction responsible was synthesis of enzymes involved in N 0 reduction, then 2 this pattern might also be observed in bacterial culture. This was tested using a Flavobacterium sp. and the results are presented in Figure 16 and Table 17. The cells were grown aerobically and then exposed to the anaerobic assay conditions. In the absence of N03- it was possible to measure the rate of N20 reduction directly. Two rates 79 .m8mmo oo8uoHom o co omoooaw NH.0 m 0N.0 800.00 H.0m8 Amm.0v 0.0m om-mq upon 08 N8.0 A00.80 0.088 A00.Hv m.mm qmlmN 8m.0 Amo.0v 0.m08 Amm.0v m.mm wNImN Moo; 8N 86888588866 o«z 888.88 «.88 A8«.ov «.8- 88-88 86888588868 o«z 888.88 8.«8 A88.ov 8.8- 88-88 m8.0 A00.00 0.08 Acw.0v N.N mNInN 88.0 Amm.0v 8.0m A00.0v 8.8m NHIm 8m.0 A00.80 m.am A00.Hv 0.0N qu moon N IIIIA Moos. afiom w.mmw moaoachIIII Amazonv um oooom o 2 mm HI I N Cowman 0889 omoooaw NH.0 ooaoouo Hauou o08uOOH08888ooo oO8uoavoua 0 z . 8 mo cowuuoaoum mo mum“ mo moon uoz coaumo8m8888oop pom :08uoooouo 0 N .H8om caumxooum m a« 2 00 money Ozu co cowufioom Omooaaw mo uoommo OLH .0H OHan N20 ((1 HOLES) 8O DENITRIFICATION AND N20 REDUCTION IN A FLAVOBACTERIUM SP. 30 - DENITRIFICATION 40- (+c H ) TCHL’“ 2 2 . / + CHL / - 30— 2° N20 REDUCTION - +CHI? 'o_ l/\ ‘///; -+(HIE; ' I I I 1. 5L1 l O 2 4 6 8 30 32 TIME (hours) Figure 16. N20 reduction and denitrification with time in a Elaxebacteriqm sp. 81 .8«.8-«.ov 868666866 8 N HOuOu mo a08uuoaoum: ou oanoumoaoo % macfia 00.H Amuoxomun n80 cam .oOHuoovoua muOoHHm z + O .QHIHH oHnoH C8 :0 oum muoxoouo a8 mooHo> one z mo ouom\cowuoaoou o N N 2 mo uosoouo .08uou 885u 2 How cone nowuoO8m88u8ooo mo ouou onu oo8auoooo ou A0.mlm.Nv now: mos vowuoa oE8u HouoH m .aowuoowmwuu8aoo mo oumu onu :8 on :Halooa Hofiu8a8 ou one 2 86 868m 86 68888 6 n .888> poo oHHoo mo onw8o3 880 we an o 888.8L «8.8 A00.Hv m.mm Am0.0v H.Nm NNIom + 888.8. 88.8 888.88 8.8« 888.88 8.88 «.8-«.8 + Aw.wlm.Nv 888.8_ 88.8 888.80 8.«8 A«8.88 8.8« 8.8-8.8 + 88«.o_ 88.8 A88.ov 8.88 Aoo.80 8.88 «8-88 - Hom.0_ 0m.0 Amm.0v 0.80 A00.0v 0.08 N.wIN.0 I A0.mlm.Nv ammo.0H Nm.0 Ao0.80 0.mw A00.00 0.0N pN.m-.N.0 I ANz + 0sz mo aofiuoaoouo mo ouom IA Moon. oHHoo wa.wmw moaoeovl Aouaonv HOOHGocaamuoano HI NI N N ooauoa oa8H 0 2 mo oo8uoooou mo ouom :OHuMOHmwuuwooo coauoovou 0 2 mo ouom mo ouom .Houwoocoaouoano mo oooomno.ooo oosomoua onu o8 o.am aofiuoooono>oam o :8 mumownouoooo mo uowoo uoumo cowuo08MNHuwooo one GOHOODvoH oNz mo mouom .NH oHnoH 82 were determined in the presence and absence of chloramphenicol: the rate of denitrification (N20 production in the presence of CZHZ) and N20 reduction. In the presence of chloramphenicol the rate of N20 reduction remained relatively constant throughout the 32—hour assay. In the absence of chloramphenicol, the rate of N20 reduction increased signifi- cantly after about 3 hours. This indicates dg'ngvg synthesis of proteins involved in N20 reduction. The denitrification activity exhibited an initial lag (l to 2 hours) after which linear rates were established. In the presence of chloramphenicol the rate of denitrification was slightly lower (after 5 hours) than in its absence. The pattern of .activity shown in these data is consistent with the previously reported activity patterns in soils. After a period of anaerobic incubation, a significant increase in N20 reduction activity occurred; a comparable increase in the rate of N 0 production did not occur. Other resting 2 cells were incubated anaerobically in the absence of N 0 and N0 -. At 2 3 29 hours, N20 was added and the rate of its reduction was found to be 17.6 nmoles gas-mgml-hourn1 (29 to 31 hours). This rate was lower than the rate of N20 reduction for this period in the presence of chloramphen- icol (32.1 nmoles gas-mg-l-hour-l). Hence, the presence of N03- or N20 was required to produce the increase in N20 reducing activity. This result is similar to that obtained in soil. DISCUSSION In the three soils investigated, a consistent pattern of N20 and N2 production was found, which corresponds to length of anaerobic period. After an initial period of anaerobiosis (from 12 to 28 hours) the proportion of the gaseous products of denitrification occurring as 83 N20 sharply decreased. The time required for this to happen and the magnitude of the decrease varied with soil and time of collection; but the time and magnitude of this shift were relatively constant over repeated experiments if the same soil from the same collection date was used. To my knowledge, the distinct changes that occur in the ratio of N20/(N20 + N2) with time, have not been previously noted in the literature. But on examination of reported data (2, 3), the pattern that I have seen is apparent. For example, in the recent paper by Freney g£_gl. (3), nitrous oxide evolution from a field soil was followed for 22 days. With water contents adjusted at the outset to field capacity or above, the soil served as a source of nitrous oxide for the first 1 to 2 days after which the soil became a net N20 sink. The work reported here suggests that at least two factors are involved in the decrease in the ratio of N20/(N20 + N2) after a period of anaerobiosis: (i) dg_gggg enzyme synthesis and (ii) carbon depletion. The sequence of synthesis of denitrifying enzymes after the onset of anaerobic conditions has been investigated to a limited extent in bacterial cultures. Payne and Riley (9) have reported that all of the enzymes involved in denitrification were present after 40 min of anaerobio- sis in Pseudomonas perfectomarinus. But these investigators did not determine the relative activities of the specific enzymes at 40 min or what happened during longer periods of anaerobiosis. Matsubara (7), working with Pseudomonas denitrificans grown under low aeration condi- tions, reported that the cells were already induced for N 0 reduction 2 when shifted to anaerobic denitrifying conditions. He also reported that cells grown anaerobically in the presence of N 0 had more N O 2 2 84 reducing activity than cells grown anaerobically in the presence of NO3-. In later experiments with §;_perfectomarinus, Payne g£_§l, (10) found that the production of all of the denitrifying enzymes was dere- pressed by anoxia or lowered O2 tensions and that the synthesis of N20 reductase begins first and reaches a maximum first in cells shifted from aerobic to anaerobic conditions. To the limited extent to which they are applicable, these results are consistent with my observations. The presence of N20 or NO3- (possibly N20 produced from N03-) did promote increased NZO-reducing activity both in soils and in bacterial culture. In the Flavobacterium sp., I observed an increase in N O reducing 2 activity which was far greater than any subsequent increase in overall denitrification rate. Available carbon (electron donor) was the second factor which was 0/ found to be involved in the time-dependent decrease in the ratio of N2 (N20 + N2). It may be that, as the electron donor becomes limiting, the relative proportion of N20 decreases; however, I have no direct evidence to support this. What my data do indicate is that if exogenous carbon is added, the relative proportion of N20 increases. It is possible that the decrease in the ratio of NZO/(NZO + N2) was due only to an increase in N20 reducing activity. It has previously been reported that the addition of carbon to soils caused a decrease in the quantity of N20 accumulating (8, 14). This seems to be the opposite of what I have observed, however, the disparity can be explained by several factors. Nommik (8) reported the quantities of N20 and N2 that accumulated in a closed system over the lZ-day period required to deplete the added NO 3 (9.21 mg N) in the absence of added carbon. The appearance of N20 was transient, it accumulated, and later disappeared. He then added glucose 85 to an identical system and observed that the N03 was depleted in 5 days and that the total amount of N20 that accumulated (and was later reduced) was lower than in the absence of added glucose. In the presence of glucose the overall rate of denitrification was much higher than in its absence. The observation that less N20 accumulated in the presence of glucose was based on a single observation at 2 days; it is quite possible, if not probable, that a much higher quantity of N20 had accumulated between 0 and 2 days but was missed. Also, the concentration of N03- present declined much more rapidly in the presence of glucose than in its absence; this may also have affected the transient N 0 accumulation, 2 because in the presence of higher concentrations of NO3- one would expect more N 0 production relative to N 2 2' Although the soils were incubated under total anaerobiosis, which rarely occurs in most natural soils, the responses observed should be typical of the prOportion of anaerobic microsites in soil aggregates. It is now thought that anaerobic zones grow and decline in volume in response to moisture conditions (11). Thus, the changes following onset of anaerobiosis, reported here, would be expected to occur in newly created or growing anaerobic zones in soil aggregates. Due to the lack of sensitivity of the Hot Wire detector used in this research, I have not been able to investigate the distribution of products during the first two hours of anaerobiosis in soils. Our early results, however, suggested that N2 was the dominant product during this period (12); However, this observation needs to be further investigated. 63 The more sensitive, Ni electron capture detector, now available to me, should facilitate this investigation. This may well be the most important 86 period in nature, since these very short-term periods of O2 depletion are common after rainfall. 10. ll. LITERATURE CITED Balderston, W. L., B. Sherr and W. J. Payne. 1976. Blockage by acetylene of nitrous oxide reduction in Pseudomonas perfectomarinus. Appl. Environ. Microbiol. 31:504-508. Blackmer, A. M. and J. M. Bremner. 1978. Inhibitory effect of nitrate on reduction of N 0 to N by soil microorganisms. Soil Biol. Biochem. 10:187—191? 2 Freney, J. R., 0. I. Denmead and J. R. Simpson. 1978. Soil as a source or sink for atmospheric nitrous oxide. Nature 273:530-532. Ishaque, M. and M. I. H. Aleem. 1973. Intermediates of denitrifica- tion in the chemoautotroph Thiobacillus denitrificans. Arch. Microbiol. 94:269-282. Klemedtsson, L., B. H. Svensson, T. Lindberg and T. Rosswall. 1978. The use of acetylene inhibition of nitrous oxide reductase in quantifying denitrification in soils. Swedish J. of Agricultural Res. 7:179. Koike, I. and A. Hattori. 1975. Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseudomonas denitrificans under nitrate-, nitrite-, and nitrous oxide—limited conditions. J. Gen. Microbiol. 88:11-19. Matsubara, T. 1971. Studies on denitrification. XIII. Some preperties of the NZO-anaerobically grown cell. J. Biochem. 69: 991-1001. Nommik, H. 1956. Investigations on denitrification in soil. Acta Agr. Scand., 6:195-228. Payne, W. J. and P. S. Riley. 1969. Suppression by nitrate of enzymatic reduction of nitric oxide. Proc. Soc. Exp. Biol. Med. 132:258—260. Payne, W. J., P. S. Riley and C. D. Cox. 1971. Separate nitrite, nitric oxide, and nitrous oxide reducing fractions from Pseudomonas perfectomarinus. J. Bacteriol. 106:356-361. Smith, K. A. 1978. A model of the extent of anaerobic zones in aggregated soils, and its application to estimates of denitrification. Abstr. 11th International Congress of Soil Science. 1:304. 87 12. l3. 14. 15. 16. 88 Smith, M. S., M. K. Firestone and J. M. Tiedje. 1978. The acetylene inhibition method for short-term measurement of soil denitrification and its evaluation using nitrogen-13. Soil Sci. Soc. of Am. J. 42: 611-615. Terai, H. and T. Mori. 1975. Studies on phosphorylation coupled with denitrification and aerobic respiration in Pseudomonas denitrificans. Bot. Mag. (Tokyo) 88:231—244. Wijler, J. and C. C. Delwiche. 1954. Investigations on the denitrifying process in soil. Plant and Soil 5:155-169. Yoshinari, T. and R. Knowles. 1976. Acetylene inhibition of nitrous oxide reduction by denitrifying bacteria. Biochem. Biophys. Res. Comm. 69:705-710. Yoshinari, T., R. Hynes and R. Knowles. 1977. Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biol. Biochem. 9:177-183. CHAPTER IV INVESTIGATIONS ON NITRIC OXIDE AS AN INTERMEDIATE IN DENITRIFICATION In the last ten years much evidence has accumulated which supports the role of nitric oxide as an intermediate in denitrification. Working with cell-free extracts or partially purified enzyme preparations from Pseudomonas perfectomarinus, (9, 23, 24), §L_denitrificans (18, 34), g; aeruginosa (27), Thiobacillus denitrificans (12), and P; stutzeri (7, 8) investigators have identified nitrite reductase activities which produce NO, and nitric oxide reductase activities which reduce NO to N20 or N2. There is also evidence that phosphorylation occurs during NO2 reduction to N20 (12, 15, 34), but whether the energy yield is associated with the step from N02 to NO or the reaction from NO to N20 has not been con- clusively determined. Nitric oxide production from intact cells of Corynebacterium nephridii and T; denitrificans has been reported (3, 28). Recently, Pichinoty and coworkers (26) have reported the isolation of ten strains of Bacillus which may utilize NO as a terminal electron acceptor for growth. The interpretation of some of the evidence which appears to establish NO as an intermediate in denitrification is equivocal. It is known that the nitrogenous oxides interact both chemically and enzymatically with several of the artificial electron donors used in cell-free extracts and that NO can be produced from nitrite (and N from N0) by nonspecific or 2 non-enzymatic reactions (13, 18, 22, 25, 37). In addition, nonspecific binding of NO to cytochromes is known to occur (30) and reduction of NO to N20 by a nitrite reductase-cytochrome has been reported (17). Thus, 89 90 there is a question whether the reductase activites identified in crude cell-free preparations have this function in_vivo. Reduction of NO has been observed in bacteria, fungi, algae and higher plants which do not 3 it is probable that the reduction of NO is being catalyzed by an enzyme carry out dissimilatory reduction of NO (10, l4, 16). In these cases for which nitric oxide is not the normal substrate. In the literature on denitrification in soils, it has generally been concluded that the appearance of NO resulted from chemical decomposi— tion of nitrous acid (1, 2, 4, 5, 6, 20, 21, 29, 33, 36, 38). Allison reviewed the chemical reactions which may be involved (1). Under acid conditions nitrous acid decomposes to produce nitric oxide and nitric acid. In an anaerobic system the reactions would be (31): 419102 -> 2N0 + 2No2 + 21120 [1] ...) 2N02 + N204 [2] N204 + H20 + Imo3 + HNOZ [3] The combination of these equations [1, 2, 3] yields the equation commonly given for nitrous acid decomposition: 3HNO 1 HNO + 2N0 + H o 4] 2 3 2 I It has generally been found that the decomposition of nitrous acid to nitric oxide is significant only in soils of an acid pH (§_6.0) (l, 20, 21, 29, 36). Sneed and Brasted (30) give the equilibrium constant K of equation [5] as 6.0 x 10-4, HN02 I H+ + N02" [5] Thus at a pH of 6, only 0.17% of the nitrite present would exist as HNOZ. The appearance of N0 has not commonly been reported in neutral or 91 alkaline soils. Allison, in his review, concludes that in neutral or alkaline soils there would be little if any NO formation resulting from decomposition of HNO2 (1). Cady and Bartholomew (6) attempted to identify the source of NO appearing in acid soils by adding 15N03- to soil and measuring the rates of 15NO appearance in an added nonlabeled N0 pool. They concluded that the rate of 15NO appearance could not be explained on the basis of nitrous acid decomposition alone. Surprisingly, they never considered the possibility of exchange of a biological 15NO intermediate with the nonlabeled NO pool. St. John and Hollocher (32) working with P; 15 15 aeruginosa found almost no exchange of the N label added as N02- with an added pool of nonlabeled NO. These investigators concluded "that N02- and N0 are denitrified by separate pathways, at least prior to the formation of the first bi—nitrogen compound". If NO is actually an intermediate in biological denitrification then it may exist only transiently as an enzyme bound species. In the work reported here, I have attempted to investigate the role of NO as an intermediate of denitrification in pure cultures and in soils using isotOpe exchange experiments. The 13N label was added as either 13NO3- or 13N02_ and the appearance of the resulting 13NO in a pool of nonlabeled NO was determined. MATERIALS AND METHODS .§§cteria1 strains; culture and preparation Four strains of denitrifiers were used which had been previously isolated from soils by T. N. Gamble. These denitrifiers were chosen as rEpresentative strains from a numerical taxonomic study of 147 isolates 92 from soils, sediments and oxidized poultry manure (11). The strains used were: §;_fluorescens 72; Flavobacterium sp. 175; Alcaligenes facaelis 191; and P; aeruginosa 156. These organisms were grown and harvested as previously described in Chapter I. In addition several vials °f.2; fluorescens and Flavo- bacterium were autoclaved for 15 min and capped with sterile Hungate septa and aluminum crimp seals. The gassing manifold used was equipped with sterile Cathivex filters (Millipore Corp., Bedford, Mass.) and sterile needles to avoid contaminating the autoclaved solutions. All experiments were conducted in 0.02 M_phosphate buffer pH 7.0. 80113 used and preparation The soils used were a Brookston loam and a Spinks loamy sand described in Table 6 of Chapter II. The preparation of the soils was identical to that described in Chapter I. Prior to the experiment a number of soil containing flasks were autoclaved for 30 min. When desired, the pH of the soils was altered by the addition of 1 N HCl or 1 N NaOH. The pH was found to be relatively stable after about 15 min of mixing. The pH was determined in the supernatant after the soil had settled. After the experiment was complete the pH of the soils was again determined. The pH had usually changed by 0.2 to 0.4 units. The pH recorded for the soil used was the average of the values measured before and after experiment. Experimental procedure The general procedure used was very similar to that described in Chapter I. For all of the studies with NO the syringes used for addition of NO or for sampling of gas were flushed three times with helium to exclude 02 immediately prior to use. The nonlabeled NO was 93 added to each incubation vessel about 2 min prior to the addition of the 13N—labeled substrate. Samples (0.5 ml) were taken of the headspace gases after the specified time of incubation. Immediately after the samples were taken from the cultures, 0.2 m1 of nonlabeled N0 gas was added to each syringe as a mass carrier; this was required because very small quantities of NO did not elute well from the chromatographic columns. When large quantities of 13NO - were required, the untreated water 2 containing the 13NO3-, 13NO - and 13NH + was passed through a 2 cm 2 4 cadmium column. The isotOpe solution was then dried, resuspended in 100 pl of H l3N0 20 and injected into the HPLC. The fractions containing the 2- were collected and used for experimentation. The procedures for data acquisition were the same as that described in Chapter I. RESULTS Soils 13N03- was added to anaerobic slurries of a Brookston and a Spinks soil. If an NO intermediate occurred during the reduction of N03 to N2 then it would have been l3N labeled. The trapping of 13NO in an added pool of nonlabeled NO is reported in Table 18. In the Brookston soil, as the concentration of added NO was increased, the quantity of 13NO trapped in the NO pool increased. With the addition of 0.16 atm 13 13 13 of NO, 96% of the N labeled gas appeared as NO. NO was also found in an autoclaved Spinks soil but at only one-tenth the quantity of that found for the non-autoclaved soil. This indicates that the production of 13NO was largely biologically mediated. However, it was 94 Table 18. Trapping of 1:NO in a nonlabeled NO pool in Brookston and Spinks soils. NO added 13N0 [13N]-N20 [13N]-N2 7 13N -2 2 b o ~gaS (10 atm) -------- 10 disintegrations - ------- as NO Brookstonc 0 3.2 (0.8) 11.6 (1.0) 766.8 (7.4) 0.4 8 162.2 (2.2) 5.5 (0.4) 3.2 (0.4) 94.9 16 395.6 (4.3) 2.2 (0.4) 13.0 (0.8) 96.3 c Spinks 14 264.0 (2.7) 11.3 (0.8) 29.0 (1.0) 86.8 Spinks- autoclaved 14 29.3 (0.9) 0 2.4 (0.5) 92.4 a 13 - N substrate was predominantly 13NO -, no carrier NO was added. 3 3 Integrated over gas peak. c Brookston soil sampled at 10 min, Spinks at 32 min. Autoclaved for 30 min 8 hours prior to experiment. 95 1 _ possible that the biological component was the reduction of 3NO to 3 l3 - 13 13 N02 with the resulting H NO2 chemically decomposing to form N0. In an attempt to resolve this point, I added purified 13N02— and 13 purified N03- to the Brookston soil. The relative composition of the 13N-gases produced was similar (Table 19); 13NO comprised 97.9% of the 13N02- derived products and 91.8% of the 13NO3- products in the presence of 0.16 atm of NO. Because large extracellular pools of 13N02~ from 13NO3- would not be expected, the similarity in gas composition for the two substrates suggests biological production and exchange of 13N0. However, results with sterile controls are required before this conclu- sion can be made. If N02- were reacting chemically as nitrous acid, then the produc- tion of NO should have been enhanced by lowering the pH of the soil. The influence of soil pH on the production of 13N0 in a Brookston soil and an autoclaved control is shown in Table 20. In the lO-min samples from the biologically active soil, there was less 13NO produced at a pH of 7.9 than at the lower pH's, but this difference had disappeared by the 40-min sampling time. At 40 min, there was no significant differ— ence in 13NO production by the three soils of pH 4.5 - 7.9. 13NO was again detected in the autoclaved soil controls but at 1/10 the level of that found in the biological system. Enhanced 13NO production was not found in the autoclaved soil of pH 4.5. These data also suggested that the 13NO production in soils was largely biologically mediated. However, recognizing that the non-living soil matrix is a highly catalytic medium, it seemed best to resolve the question of biological versus chemical 13NO production by use of bacterial cultures. 96 Table 19. Influence of source composition on 13NO appearance in Brookston soil. l3N NO added 13NO [13N]-N20 [13N]-N2 % l3N—gas Substrate (x10—2 atm) ------ 103 disintegrationsa----- as NO 13 - N02 16 92.0 0.8 1.1 97.9 13N03’b 16 108.3 8.1 1.7 91.8 13N03' 8 14.1 8.8 2.1 56.4 a Integrated over peak area. b About 34.7 uCi 13NO - and 17.2 uCi 13NO - injected. No carrier N was added. Samples taken after 10 min incubation. 97 Table 20. Influence of soil pH on 13NO appearance in an autoclaved and nonautoclaved Brookston soil . Time of 13NO [13N]-N 0 [13N]-N % lBN-gas sample 2 2 b 2 (min) Soil pH ------- 10 disintegrations --------- as NO c Brookston 10 4.5 425.6 (2.3) 2.1 (0.3) 28.7 (0.7) 93.3 10 6.2 526.6 (2.4) 19.6 (0.5) 14.1 (0.4) 94.0 10 7.9 169.5 (1.7) 5.9 (0.4) 26.1 (0.8) 84.1 40 4.5 602.9 (3.1) 10.8 (0.6) 42.8 (0.9) 91.8 40 6.2 533.6 (3.6) 7.4 (0.8) 52.9 (1.3) 89.8 40 7.9 639.4 (3.6) 19.3 (1.0) 56.6 (1.4) 89.4 Autoclaved,1 Brookstoncu 30 4.5 40.7 (1.1) O 2.3 (0.3) 94.7 30 6.2 40.7 (1.0) 0 2.2 (0.3) 95.0 30 7.9 67.4 (1.0) 0 2.5 (0.3) 96.4 a 13 - N substrate was predominantly 13NO3_, no carrier N03 was added. b Integrated over peak area. c Nonautocliged soil received 56.6 uCi 13N; autoclaved soil received 41.7 uCi N. All soils received 0.12 atm of NO. Autoclaved for 30 min 8 hours prior to experiment. 98 .cfia on no nmxmu moaaamm mow n .mumnumnomIz saws swoon mmB nmwupoo ZIImoz w: H .INozmH NOH uaonm £883 ImOZMH 8Husmafiaonoua was oumuummmm ZmH m m.mm N.Nw Am.ov m.mq AH.ov N.H a¢.oV N.Nm m.¢ o.w 8.888 AN.ov w.m Am.ov N.oN A8.ov q.wm o.m m.o 8.888 Ao.ov m.o An.ov H.8HH AH.ov m.N m.H o m.waa o Aq.ov m.wHH no 0 oz mm IIIIIIIIIIIIII IIIIImoo8umuw8888888 moHIIIIIIIIII II 8888 NIoHV mowI . mam muo N I N I m m 288 a 288 8 a 8288 z H288_ 8 z ~288_ 8 88 oz .02 swoon mo moowumuunmoaoo mo8mum> nu83 mcoommuoaam mmcoeooaomm :8 moz soum :OHuomnouo oz .HN magma MI ma ma .am So8uouomno>m8m on 0» oooom mm3 ozu £883 oooom mm: um8uumo ZII oz w: 8 uoon< N 8o8uumo oz .mCoommuoo8w 4M ocu pom oz M8 3 .o8aaom zoom 08 oooom mos 02 Sum no.0 m 99 m.mm AN.NV w.8w on o.o on 0.0 m8 : 8.wm 88.08 m.om AN.ov N.8 88.0v m.o m oo>m8oou=oI o.M8m Am.mv m.omN 88.8V 8.8 Am.8v 0.08 m8 : 8.m8m Am.NV N.8Nm Am.ov m.N Ao.ov «.m8 m n.om a=8uouoono>o8m m.w8N Am.mV m.m8N on 0.0 ADV 0.0 on : N.O8N 88.8v w.NON 8N.ov w.N on 8.0 08 oo>m8oou=mI 8.088 AN.NV 8.80 Am.NV 8.0m Am.mv o.me om : N.Nnn Ao.8v N.oo8 Ao.8v N.N8 a8.mV m.8mo O8 nmaoomonoa8m dmw mnowuouwmuGHmHo N08 I AQHEV ouooauooue 888 288 88888 8288 82-8288_ 882-82888 8888 . Noz :u83 ooumnooa8 .qm aa8umuomno>o8m ooo mnoommuoa8m mocoaoooomm mo mno8uouonoua o8no8> mam mM8uoum a8 :O8uoaoou onm c08uuaooua oz .NN o8nmy m8 100 Bacterial culture 13NO production by several denitrifying cultures is shown in Tables 21 and 22. 13N03- and nonlabeled N0 at several concentrations were added to §;_fluorescens cells. It can be seen in Table 21, that as the size of the added NO pool increased, the amount of 13NO produced increased. There may have been some NO toxicity to the cells, as the total 13N gas production declined with increasing NO additions. This experiment was also done with Flavobacterium and similar results were obtained. The most valid control for chemical decomposition of HNO2 was to add 13N02- to autoclaved and viable cells (Table 22). In both the 10 and 30 min samples from P; fluorescens, more 13NO was detected in the sterile control than in the viable cells. For the Flavobacterium sp., significant quantities of 13N0 were again detected in the sterile control but in quantities that were significantly less than those found for viable cells. In the viable Flavobacterium, most of the 13N gas 1 produced was as 13NO (95% as 3NO); while in the §;_fluorescens only 7 to 13% of the 13N gas was 13NO. Although this difference may be do to the fact that they are different bacteria, it could also be related to the addition of 1 pg N02--N carrier to the P; fluorescens and the absence of added carrier in the Flavobacterium sp. These cell suspen- sions were buffered at pH 7.0. It is apparent that there was signifi- cant chemical decomposition of 13N02- to form 13N0. But the data 13 13 12 indicate biological reduction of NO to form [ N]-N20 and [ N]—N2. 13 The quantity of N0 in the viable P;_fluorescens-declined between the 10 and 30 min sampling, while the amount of [13N]-N20 and [13N1-N2 detected increased. Biological reduction of NO to N 0 and N2 was 2 101 confirmed by the microthermistor detector analysis of the quantities of gases present at different times. In both the presence and absence of added carrier N02- or N03-, the quantity of nonlabeled NO decreased with time and the quantity of nonlabeled N 0 increased with time (data 2 not presented). DISCUSSION The data on 13NO production in soils seemed to indicate that the NO resulted from biological denitrification. The appearance of 13NO was not enhanced in soils in which the bulk pH had been lowered to 4.5, suggesting that chemical decomposition of HNO2 was not the dominant driving force. But the chemistry of HNO2 in soil is exceedingly complex (35) since its decomposition can be promoted by metallic cations (39,40), and organic matter (20). HNO2 is also known to react with aliphatic amino groups (1) and aromatic compounds (33). Hence, it is unwise to base any conclusions on the role of NO in denitrification, on data obtained from soils alone. Work with autoclaved bacterial culture preparations indicated that a chemical mechanism was involved in the production of 13NO; although it is possible that biological denitrification is responsible for a portion of the 13NO production in the viable cells. The data reported here do not elucidate the role of nitric oxide in denitrification, but reflect the difficulty of doing definative work with this reactive gas. 10. 11. LITERATURE CITED Allison, F. E. 1963. Losses of gaseous nitrogen from sails by chemical mechanisms involving nitrous acid and nitrites. Soil Sci. 96:404-409. Bailey, L. D. 1976. Effects of temperature and root on denitrifi- cation in a soil. Can. J. Soil Sci. 56:79-87. Baldensperger, J. and J. L. Garcia. 1975. Reduction of oxidized inorganic nitrogen compounds by a new strain of Thiobacillus denitrificans. Arch. Microbiol. 103:31-36. Ballag, J. M., S. Drzymala and L. T. Kardas. 1973. Biological versus chemical nitrite decomposition in soil. Soil Sci. 116:44- 50. Cady, F. B. and W. V. Bartholemew. 1960. Sequential products of anaerobic denitrification in Norfolk soil material. Soil Sci. Soc. Amer. Proc., 24:477-482. Cady, F. B. and W. V. Bartholomew. 1963. Investigations of nitric oxide reactions in soils. Soil Sci. Soc. Amer. Proc. Chung, C. W. and V. A. Najjar. 1956. Cofactor requirements for enzymatic denitrification I. Nitrite reductase. J. Biol. Chem., 218:617-626. Chung, C. W. and V. A. Najjar. 1956. Cofactor requirements for enzymatic denitrification II. Nitric oxide reductase. J. Biol. Chem. 218:627. Cox, C. D. and W. J. Payne. 1973. Separation of soluble denitri- fying enzymes and cytochromes from Pseudomonas perfectomarinus. Can. J. Microbiol. 19:861-872. Fewsan, C. A. and D. J. 0. Nicholas. 1961. Nitric oxide reductase from Pseudomonas aeruginosa. Biochem. J. 78:9P. Gamble, T. N., M. R. Betlach and J. M. Tiedje. 1977. Numerically dominant denitrifying bacteria from world sails. Appl. Environ. Microbiol. 33:926-939. 102 12. l3. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 103 Ishaque, M. and M. I. H. Aleem. 1973. Intermediates of denitri- fication in the chemoautotroph Thiobacillus denitrificans. Arch. Microbiol. 94:269-282. Iwasaki, H., S. Shidara, H. Suzuki and T. Mori. 1963. Studies on denitrification. VII. Further purification and prOperties of denitrifying enzyme. J. Biochem 53:299-303. Kemp, J. D. and D. E. Atkinson. 1966. Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinu- cleotide. J. Bacterial. 92:628-634. Koike, I. and A. Hattori. 1975. Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseudomonas denitrificans under nitrate-, nitrite-, and nitrous oxide-limited conditions. J. Gen. Microbiol. 88:11-19. Lazzarini, R. A. and D. E. Atkinson. 1961. A triphasphapyridine nucleotide-specific nitrite reductase from Escherichia coli. J. Biol. Chem. 236:3330—3335. Matsubara, T. and H. Iwasaki. 1972. Nitric oxide-reducing activity of Alcaligenes faecalis cytochrome cd. J. Biochem. 72:57- 64. Miyata, M. and T. Mori. 1968. Studies on denitrification VIII. Production of nitric oxide by denitrifying reaction in the presence of tetramethyl-p- phenylenediamine. J. Biochem. 64:849-861. Miyata, M., T. Matsubara and T. Mori. 1969. Studies on denitrifi- cation XI. Some prOperties of nitric oxide reductase. J. Biochem. 66:759-765. Nelson, D. W. and J. M. Bremner. 1969. Factors affecting chemical transformations of nitrite in soils. Soil Biol. Biochem. 1:229- 239. Nommik, H. 1956. Investigations on denitrification in soil. Acta Agr. Scand., 6:195-228. Payne, W. J. 1973. Reduction of nitrogenous oxides by micro- organisms. Bacteriol. Rev. 37:409-452. Payne, W. J. and P. S. Riley. 1969. Suppression by nitrate of enzymatic reduction of nitric oxide. Proc. Soc. Exp. Biol. Med. 132:258-260. Payne, W. J., P. S. Riley and C. D. Cox. 1971. Separate nitrite, nitric oxide, and nitrous oxide reducing fractions from Pseudomonas perfectomarinus. J. Bacteriol. 106:356-361. Pichinoty, F. 1969. La denitrification bacterienne. I. Utilization des amines aromatique comme donneuses d'electrons dans la reduction du nitrite. Arch. Mikrobiol. 69-314-329. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 104 Pichinoty, F., J. L. Garcia, M. Mandel, C. Job, and M. Durand. 1978. Isolation of bacteria that use nitric oxide as a respira- tory electron acceptor in anaerobiosis. Comptes Rendus - Sciences Naturelle 286:1403. Rowe, J. J., B. F. Sherr, W. J. Payne and R. G. Eagon. 1977. A unique nitric oxide-binding complex formed by denitrifying Pseudomonas aeruginosa. Biochem. BiOphys. Res. Commun. 77:253. Renner, E. D. and G. E. Becker. 1970. Production of nitric oxide and nitrous oxide during denitrification by Corynebacterium nephridii. J. Bacteriol. 101:821-826. Reuss, J. D. and R. L. Smith. 1965. Chemical reactions of nitrites in acid soil. Soil Sci. Soc. Amer. Proc. 29:267-270. Sherr, B. F., J. J. Rowe, G. M. King, W. Zumft, and W. J. Payne. 1978. Partial purification and characterization of the nitric oxide reductase complex of Paracoccus denitrificans. Abstr. Annu. Meet. Am. Soc. Microbial. K20. p. 130. Sneed, M. C. and R. C. Brasted. 1956. Comprehensive inorganic chemistry. V. D. Van Nostrand Co., Inc. Princeton, NJ. St. John, R. T. and T. C. Hollocher. 1977. Nitrogen 15 tracer studies on the pathway of denitrification in Pseudomonas aeruginosa. Stevenson, F. J., R. M. Harrison, R. Wetselar, and R. A. Leeper. 1970. Nitrosation of soil organic matter: III. Nature of gases produced by reaction of nitrite with lignins, humic substances, and phenolic constituents under neutral and slightly alkaline con- ditions. Soil Sci. Soc. Amer. Proc. 34:430-435. Terai H. and T. Mori. 1975. Studies on phosphorylation coupled with denitrification and aerobic respiration in Pseudomonas denitrificans. Bot. Mag. (Tokyo) 88:231-244. Van Cleemput, O. and L. Baert. 1976. Theoretical consideration of nitrite self-decomposition reactions in soil. Soil Sci. Soc. Amer. J. 40:322-324. Van Cleemput, 0., W. H. Patrick and R. C. McIlhenny. 1976. Nitrite decomposition in flooded soil under different pH and redox potential conditions. Soil Sci. Soc. Amer. J. 40;55-59. Verhoeven, W. 1956. Studies on true dissimilatory nitrate reduction: V. Nitric oxide production and consumption by micro- organisms. Antonie Van Leeuevenhoek 22:385-406. Wijler, J. and C. C. Delwiche. 1954. Investigations on the denitrifying process in soil. Plant and Soil 5:155-169. 39. 40. 105 Wullstein, L. H. and C. M. Gilmour. 1964. loss of nitrite from clay and soil systems. Wullstein, L. H. and C. M. Gilmour. 1966. tion of nitrogen gas. Nature 210:1150-1151. Non-enzymatic gaseous Non-enzymatic forma- APPENDIX The methods reported in this appendix have been developed over a period of two years by a number of people. Richard Firestone, Michael Betlach, Scott Smith, James Tiedje and Bill Caskey have all contributed significantly to the develOpment and testing of these methods. Much of the information reported here is included in a paper submitted to Soil Sci. Soc. Amer. J. This appendix does, however, contain details on the configuration of the detection system which does not appear elsewhere. APPENDIX METHODS FOR THE PRODUCTION AND USE OF 13N IN STUDIES OF DENITRIFICATION 13N is the longest lived radioactive isotOpe of nitrogen; it has been used in the medical sciences (2, 11) and for studies of the metabolism of N2, N114+ and N03- (8, 9, 10, 15). Recently its applica- tion to studies of denitrification was demonstrated by Gersberg, g£_al, (4). The ease of use of 13N for denitrification studies is enhanced by the direct production of 13NO3-, which occurs when an H20 target is used. This was first demonstrated by LathrOp g£_gl. (7) and Gelbard gt “a1. (3) in 1973 and has been successfully used in our studies. 13N has a half-life of 9.96 min. It decays to 13C, emitting up to 1.19 MeV positrons which annihilate forming two antiparallel 0.511 MeV y-rays. The presence of both beta and gamma emission allows detection of whichever is advantageous. In this Appendix, I report on the procedures for production and purification of 13NO3- and 13N02- and on rapid automated methods for measuring [13N]—N2, [13Nl-N20, 13N0, 13NH4+, 13N02- and 13N03-. ISOTOPE PRODUCTION AND PURIFICATION Because of its short half-life, the 13N was produced at the time and site of the experiment. We bombarded water targets with proton beams from the Michigan State University sector-focused cyclotron to produce the nuclear reaction 160 (p,a) 13N. 'We used either 12.5 or 25 MeV proton beams with beam currents varying from 0.7 to 3.0 uA. The beam current employed depended on the operating efficiency of the 106 107 cyclotron that day. When the 25 MeV proton beam was used, the proton energy was degraded to about 15 MeV by inserting a 75 mil A1 adsorber into the beam line. Proton energies of between 12 and 15 MeV were desirable, to make use of the high cross-sectional area of the 160(p,o)13N reaction, while minimizing the 18O(p,n)18F, l6O(P,3p3n)11C, and 160(p,2n)150 reactions which could also occur (5). The target was deionized, glass-distilled H20 contained in a well in an aluminum block (Figure 17) which was transported to and from the beam line in a pneumatic carrier ("rabbit") (the design and aperation of this system can be found in Kosanke, ref 6). Aluminum was used as the target holder since little long-lived radioactivity is produced in this metal by the beam (2). The target well was 16.5 mm in diameter and 4.4 mm deep and holds 0.95 ml of water. Two-mil Al foil pressed against a silicon rubber o-ring by a circular A1 plate served as the beam window when the cyclotron was operating at 12.5 MeV. The back of the well was 75-mil A1 and could serve as the beam window when the cyclotron is Operating at 25 MeV. Irradiations were typically 10 to 12 min. The maximum power on the rabbit was 75 watts. The yield varied from 2 to 16 mCi of 13N depending upon beam current and energy. The target was cooled during irradiation by compressed air. No leakage of water or gases from the target was noted. After irradiation the rabbit was retrieved (5 sec) and allowed to thermally cool for l min, during which very short-lived isotopes decayed. The water was removed by puncturing the Al fail with a syringe needle and withdrawing the 13N containing water into a 1-ml disposable syringe and then transferred to a 25-ml conical boiling flask. 108 Figure 17. "Rabbit" used to contain and transport water to the beam line. 109 Besides 13N, the only other important radionuclide produced under our irradiation conditions was 18F; the presence and/or removal of this contaminating isotope will be discussed further. Table 23. 13N species produced from water target under our bombardment conditions. Ion distribution represents ranges encountered over many experiments. Ions by HPLC Cases by cc-Pca 13N-species % 13N-species % NO3 75-90 N2 90 N02 5-10 N20 10 NH4+ 0.5-25 NO NDb a 13 N gas products are minor relative to ions. None detected. Typically, a mixture of 13N chemical species was produced. The percentage of each component found is shown in Table 23. 13NO - was 3 13 + 13 - always the dominant species although some NH4 and NO were 2 usually present. To remove volatile labeled components and 13NH4+, 0.1 m1 of 10 mM NaOH was added, and the irradiated water sample was evaporated to dryness twice using a modified Buchler rotary flash evaporator. The flask was a conical 25 m1 boiling flask which rotated in a 70° C water bath behind a lead shield in a hood. The system was Optimized for rapid concentration without bumping and loss of sample. A l to 5-m1 aqueous sample could be evaporated to dryness in less than 3 min. The tip on the conical flask facilitated rapid recovery of the concentrated sample by pipet or syringe. When the evaporation was complete, 0.1 ml 110 of 10 mM HCl was added along with the desired quantity of H 0 which 2 could contain N03- or N02- as carrier. When a quantity of 13N02-, greater than that directly produced by bombardment, was desired, the following procedure was employed. The irradiated water sample, with or without 1 ug NO3--N carrier, was passed through a 0.5 x 2.0 cm Cd/Cu column (15) and then partially purified using the evaporation procedure previously described. The sample was resuspended in 300 pl of H O, and 100 pl injected onto the 2 HPLC (described in a following section). The eluted fractions contain- ing 13N02- were collected and used for experiments. ANALYTICAL PROCEDURES Gas stripping;gystem The continuous production of both [13N]-N20 and [13N]-N2 from soil slurries was measured using the differential trapping, gas stripping system diagramed in Figure 18 and briefly described previously (11, 12). It was patterned after the system used by Gersberg £5 21. (4) with two changes--both 13N-labeled gases separately rather than the sum of 13 N-labeled gases were measured, and the geometry of the traps used allowed more precise and efficient quantitation. Soil slurries (generally 75 g soil + 50 ml water) were contained in sealed 125-ml Erlenmeyer flasks. Typically 0.5-6.0 mCi 13N (0.03 to 0.3 pg N) was added to the slurry. 14N was added if desired. The soil particles were kept suspended by continuous stirring with a magnetic stirring bar. Product gases were removed by a constant flow of helium through a submerged gas sparger. The gases exiting the flask passed 111 Plotter Vii Flowmeter -—-. Compute FmeeNr '3N20 a" '3“ Drierite Added Sdl 5;;;/<;/j;;/I 2222;;ZZ/I Slurry Lead . Lead Molecular Shielding ; Shielding SIeve M I I '.\./A/, . Diagram of gas stripping system used to continuously monitor [13N]-N20 and [13Nl—N2. Figure 18. 112 through Drierite to remove water (no label was retained) and then to the N20 trap. This trap consisted of a 5.2 cm diameter flat coil of 3.2 mm OD Al tubing immersed in liquid nitrogen. Remaining gases then passed to the N2 trap which consisted of a 6.5 cm diameter flat coil of 6.4 mm OD Cu tubing packed with Molecular Sieve 13X and immersed in liquid N2. The flow rate monitored at the exit was typically 90 ml/min. Connecting tubing was 3.2 mm OD diameter silicon rubber tubing to minimize volume. Nominal gas residence time in the system was 30 sec. Data obtained using this system are shown in Figures 5 to 10. We determined that the only radioactive isotope present in these traps was 13N; no 18F-containing gases were ever detected. The y—ray detectors were 7.6 x 7.6 cm Bicron NaI(Tl) crystals (Bicron Corp., Newbury, OH) placed adjacent to the Dewars containing the traps. The face of the detector was positioned directly opposite and parallel to a coil trap. Absolute calibration was done with a 22Na source taped to the front and then the back of each coil, with the mean value used. Calibration was done each day that the apparatus was used. The NaI(Tl) crystals were housed on photomultiplier tubes connected to an ORTEC 456 High Voltage Power Supply (+1500v, ORTEC, Oak Ridge, TN) as diagrammed in Figure 19. The analog output of the photomulti- pliers was fed into an ORTEC 924 Quad Discriminator, which converted the varying amplitude voltage signals into fast negative logic pulses. A minimum threshold, above the PM noise, was set on the discriminators to reduce background. The fast negative pulses from the discriminators were fed into CAMAC, EG&G S424B scalers (E680, Oak Ridge, TN). The scalers were read by a PDP 11/45 Computer (Digital Equipment Corp., 113 .muoucaoo 8803 ocu one aoummm wcwoa8uum now 058 :u83 oom: ououoouoo zmIA8Hv8oz you co8uou=w8mnoo 08nouuoo8m no} mom ~£<408an Gawumuamwwcoo o8couuoo8m .88 888888 m sea—Ail wan. - om 119 at 700-1200 lbs/in2 to achieve the desired flow rate of 2 ml/min. The pump was turned off during injection. A sample of up to 100 ul could be injected with a Hamilton syringe before back pressure limited the quantity easily injected. To prevent microbial growth, methanolzwater (1:1) was used to fill the pump and column when not in use. The column effluent passed through 1.6 mm 0D stainless steel tubing to dual 5.1 x 5.1 cm NaI(Tl) detectors mounted behind a lead shield. The two detectors were parallel (Figure 23) and faced a coil of the effluent tubing. Depending on the activity of the sample, 100ps of the coil were varied so that the counting rate would be within the counting range of the detector. The efficiency of this counter is nominally 10%. After counting, the sample passed back across a lead shield to a fraction collector where the separated components were collected in tubes for mass analysis and/or for use as purified 13N sources. A chromatogram of the separation of the 13N ions is shown in Figure 24. 18F did not elute from the anion exchange column employed. The electronics employed with the HPLC are diagrammed in Figure 25. The signals from the NaI-PM detectors were handled either in a coincidence mode or as a single detector. Coincidence counting was used to lower background noise when sufficient activity was present. Coincidence counting can be employed because every positron annihilation event produces two antiparallel 0.511 MeV y-rays. The electronic configuration employed requires that two y-rays be detected by the two NaI detectors within a 100 nsec period for the event to be counted. The analog outputs from the two NaI-PM detectors were fed into ORTEC 417 Fast Discriminators which converted the signal to a fast negative logic pulse. The pulse from one discriminator was fed directly into an 120 HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION AND GAMMA DETECTION SYSTEM FOR I3N IONS Pressure Phosphate Gang. 80..., InjecIIon Pofl‘T-t . 77277737 """" -:"/,//.. N ' 1 ' . 'Zl- 2.“. n, . 1,171 ”1.. A P|°".' II“ I " ~ WU" fl 8 , ., ., ocquound SAX CONET ,1 _.;‘7 7,2; , .' .7 . .. :51 h. I. . _,/ ,' . ; .., v .4 Conechons / ' I ,, _ I I'-' K ’V’ val/{937.2, 1‘ . ‘ 7", v’, /". I'. - ’ / ' . If" - 5": LOGO 0 3 7.3, , ,I/ ‘ g ,. .0. 'r,;‘,I-,.' ; Shle'dlflq ~ :- 4 e I./’.. ,I, x; I/,_ . 9 - U u '/ 1., r ' U a m .- ;.,'/I"_,‘/.", / c U o rflnj’. V/x/ - 2: C . ///.' 71.0."; .l , 8 ‘) 'lOC'IDN I"; {4f ’. , ' . . .4 Comctoc :-.,/';.'/'.: '2 ‘ I, /l//, I ‘ Nol - PM Receidec Detecion Figure 23. Diagram of high pressure liquid chromatograph (HPLC) + system used to separate, quantitate, and collect 13NH4 , 13 — l3 - NO2 , and N03 . 121 HPLC SEPARATION OF ”N IONS HPLC CJ 2 20000 - a C:) .— 3 NO. (I) 15000 - ‘ II LlJ 0. (f) 10000 ~ ‘ r.— Z :I 8 5000 ' NH4+ ‘ AND; (J l .1 [\E-J_JEE::==-fi— 4- 100 300 500 TIME[SECONDS] Figure 24. Example of separation of 13NH4+, 13N02: and 13NO3- by HPLC and detected by coincidence counting of annihilation gamma rays. 122 .Eoumzm A8HvaZI08m: £883 oozo8an co8umuow8wcoo O8couuoo8m .mN onow8m >009 )1 mail. ($8,118,188 _ n 11*! o .88. t 5< 123 ORTEC 437 Time to Pulse Height Converter (TPHC), and used to start a timer. The pulse from the second discriminator was fed into an ORTEC 425 Delay, which delayed the pulse about 25 nsec. This pulse proceeded to the TPHC and stopped the timer. A voltage pulse of amplitude prOportional to the time elapsed between the start and stOp signals was produced whenever two pulses within the 100 nsec resolving time were detected. This pulse was sent to an ORTEC 420A TSCA which provided a logic signal for an ORTEC 418A coincidence unit. When the apprOpriate input was enabled, the coincidence box supplied logic pulses for the multiscale input of the Sigma 7 Computer and an ORTEC 441 Ratemeter. As described for the GC-PC, the 100 mV-fullscale output of the rate- meter was pictured on a strip chart recorder. When background noise was low and the 13N activity was expected to be low, a slow positive logic pulse was taken directly from the discriminator to the ORTEC 418A Universal Coincidence box, in which case the other input was enabled. DATA ACQUISITION AND ANALYSIS Data were recorded using both the Sigma 7 Computer and the PDP 11/45 Computer. When GC-PC or HPLC data were recorded by the Sigma 7, a standard data taking program, AUTO, was employed. AUTO recorded the events for each 2 sec interval. When flushing apparatus, GC-PC, or HPLC data were taken using the PDP 11/45, a program written by M. Betlach was employed. This program recorded data accumulated over preset arbitrary time intervals, commonly l min for the flushing apparatus and 2 sec for the GC—PC and HPLC. This program could perform on-line background, detector efficiency and half-life corrections. On- line data correction was usually employed for the flushing apparatus. W 124 When an experiment was complete a hard copy of the data was obtained from a line-printer, and if the Sigma 7 had been used for data taking, a plot of the data taken with time could be immediately obtained from a platter. For offline data correction a program written by R. Firestone, FASTFIT, was employed. This program was used for analysis of GC-PC and HPLC data; it performed background subtraction, half-life correc tion and peak integration. The zero time to which the data were corrected for isotOpe decay was an arbitrary time decided on by the experimenter. IT' I routinely employed a zero time that preceded my first data peak by about 2 min. If the experimenter did not provide a specific zero-time to the FASTFIT program, it defaulted to a zero-time which coincided with the time at which the AUTO data-taking program was initiated. FASTFIT also performed a limited statistical analysis of the data. This analysis is based on a non-linear least-squares fitting procedure traditionally employed by nuclear scientists, for analysis of counting data (1). The procedure gives an estimate of the uncertainty in the determination of the number of counts contained in a peak, due to background fluctuations, This becomes quite valuable when peak areas are small relative to background. The variance in the area of peak equals the variance of the true counting data plus the variance of the area under the background curve: 2 2 2 . aAp - aA + OAb - A + Ab The standard deviation is thus approximated by: oAP = VA + Ab 125 This assumes a linear function of background. The standard deviations routinely reported in this thesis thus provide an estimate of the error due to background noise. 10. LITERATURE CITED Bevington, P. R. 1969. Data Reduction and Error Analysis for the Physical Sciences. pp. 247-254, McGraw Hill, New York. Clark, J. C. and P. D. Buckingham. 1975. Short-lived radioactive gases for clinical use. Butterworths, London. Gelbard, A. S., T. Hara, R. S. Tilbury and J. S. Laughlin. 1973. Recent aspects of cyclotron production of medically useful radio- nuclides. In_Radiopharmaceuticals and labeled compounds, Vol. 1. Int. Atomic Energy Agency, Vienna, pp. 239-247. Gersberg, R., K. Krohn, N Peek and C. R. Goldman. 1976. Deni- trification studies with N-labeled nitrate. Science 192:1229- 1231. Kim, H. J., W. T. Milner and F. K. McGowan. 1967. Nuclear Data Tables. 3:212-215. Kosanke, K. L. 1973. A study of the helium-jet recoil-transport method. Ph.D. Thesis, Michigan State University. LathroP, K. A., P. V. Harper, B. H. Rich. R. Dinwoodie, H. Krizek, N. Lambares and I. Georia. 1973. Rapid incorporation of short- lived cyclotron-produced radionuclides into radiopharmaceuticals. In RadiOphamaceuticals and labelled compounds, Vol 1., Int. Atomic Energy Agency, Vienna, pp. 471-480. Meeks, J. C., C. P. Walk, J. Thomas, W. Lockau, P. W. Shaffer, S. M. Austin, W-S Chien,+and A. Galonsky. 1977. The pathways of assimilation of NH by the cyanobacterium, Anabaena cylindrica. J. Biol. Chem. 252:7894-7900. Meeks, J. C., C. P. Walk, N. Schilling 8&3 P. W. Shaffer. 1978. Initial organic products of fixaton of [ N] dinitrogen by root nodules of soybean (Glycine max). Plant Physiol. 61:980-983. Skokut, T., C. P. WOlk, J. Thomas, J. C. Meeks. P. W. Shaffer and W138. Chien. 1978. 1Initial organic products of assimilation of [ N] ammonium and [ N] nitrate by tobacco cells cultured on different sources of nitrogen. Plant Physiol. 62:299-304. 126 11. 12. 13. 14. 15. 16. 127 Straatman, M. G. 1977. A look at 13N and 15O in radiopharmaceu- ticals. Intl. J. Appl. Radiat. Isotop. 28:13-20. Tiedje, J. M. 1978. Denitrification in soils. In D. Schlessinger (ed) Microbiology 1978. pp. 362-366. . Tiedje, J. M., M. K. Firestone, M. S. Smith, M. R. Betlach and R. B. Firestone. 1979. 1§hort-term measurement of denitrification rates in soils using N and acetylene inhibition methods. ‘12 (ed). Proceedings Int'l Symp. on Microbial Ecology, Springer- Verlag. (In press) Tilbury, R. S. and J. R. Dahl. 197 . 13N species formed by proton irradiation of water. Radiat. Res. (submitted) Walk, C. P., J. Thomas, P. W. Shaffer, S. M. Austin, A. Galonsky. 1976. Pathway of nitrogen metabolism after fixation of N- labeled nitrogen gas by the cyanobacterium. Anabaena cylindrica. J. Biol. Chem. 251:5027-5034. Wood, E. D., F. A. J. Armstrong, and F. A. Richards. 1967. Determination of nitrate in seawater by cadmium-copper reduction to nitrite. J. Mar. Biol. Ass. U.K. 47:23-31.