THESIS Date Q..////g/7—— 0-7639 4": W; yawn-2:3 v; 1311""??? ‘..- -“-\ ‘ y r ' ‘l , _.: . &v&¢.x~‘iLcrw¢t‘. .3 xfiP ¢ . . (Stunfifl _,', «.3 mgfifiwfi 12,3232 a; “t @7‘1‘7‘Wfl‘1‘4‘7 “3*" -- 7w "9‘": an; ‘ L‘wr—s " J“ This is to certify that the thesis entitled NON-DENITRIFYING BIOLOGICAL SOURCES OF NITROUS OXIDE presented by BRUCE H . BLEAKLEY has been accepted towards fulfillment of the requirements for _M.:_S_:__._degreein Soil Science )V1531_} RETURNING MATERIALS: Piace in book drop to LJBRARJES remove this checkout from “ your record. FINES will be charged if book is returned after the date stamped be10w. NON—DENITRIFYING BIOLOGICAL SOURCES OF NITROUS OXIDE By Bruce Henry Bleakley A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1981 ABSTRACT NON-DENITRIFYING BIOLOGICAL SOURCES OF NITROUS OXIDE By Bruce Henry Bleakley Possible nonrdenitrifying sources of N20 were investigated. Microbes found to produce N20 from.NO3- but not consume it were: (i) all of the facultatively anaerobic dissimilatory reducers of nitrate to ammonium examined, Escherichia coli K12, Serratia marcescens, Klebsiella pneumoniae, Enterobacter aerogenes, Erwinia caratovora and Bacillus subtilis: (ii) a few of the assimilatory nitrate-reducing bacteria examined, e.g. Azotobacter vinelandii 12837, Azotobacter vinelandii nif-12, and Azotobacter vinelandii rifr nif-64: (iii) some but not all of the assimilatory nitrate-reducing yeasts and fungi, Hansenula sp., Rhodotorula sp., Aspergillus sp., Alternaria sp., and Fusarium sp. Neither of the two NO3--reducing obligate anaerobes examined (Clostridium KDHSZ and Vibrio succinogenes) produced N20. Production of N20 occurred only in stationary phase. The enteric bacteria and Bacillus achieved the highest conversions of N03“ to N20, reaching up to 36% of the NO3'-N recovered as NZO-N. Production of N20 was ap- parently not regulated by ammonium; enzymes produced during secondary metabolism could be the N20 source. Nitric oxide (NO) was not detected from.snteric bacteria or yeasts. N20 was also found to arise from some damaged plant tops, probably due to microbial growth. Levels of N20 above the ambient level in the atmosphere were found in human breath samples. To Mom, Dad, and Robert the pack I will always run with. 11 ACKNOWLEDGEMENTS I would like to thank Nancy Caskey, for over- seeing my first attempts at research; Gilbert Okereke, for aid in the 15N analysis; Alan Sexstone and Tim Parkin, for assistance in Operating the PE 910 gas chromatograph; Pete Cornell for aid in culturing the obligate anaerobes; and Joe Robinson for advice on the layout of some of the figures. I am also grateful for the typing ability of Cathy Hamilton, and her work on the tables. I thank Dr. James Tiedje for his guidance as my major professor. I would also like to thank the other members of my guidance committee, Dr. Frank Dazzo, Dr. Boyd Ellis, and Dr. Vernon Meints. Thanks most of all to my parents, for the sacrifices they have made for stray dogs and a graduate student. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . v LIST OF FIGURES . . . . . . . . vi INTRODUCTION AND EXPERIMENTAL OBJECTIVES . . 1 MATERIALS AND METHODS . . . . . . 3 RESULTS . . . . . . . . . . 10 DISCUSSION . . . . . . . . . 25 LITERATURE CITED . . . . . . . . 32 APPENDIX FATE OF 15NO3‘ IN Two COMPLEX MEDIA USED FOR MPN ESTIMATES OF SOIL DENITRIFIER POPULATIONS. . 37 LITERATURE CITED. . . . . 42 iv Table 10 11 LIST OF TABLES Production of nitrous oxide by several bacteria . . . . . Production of 13N gases by enteric bacteria as influenced by glucose . Reduction of 13NO and 13N02- to 13NH + by several bacteria. . . . . 4 Production of N O in early stationary phase by severai microbes in complex media containing 5 mM KNO3 . . . Production Of N O in late stationary phase by severaf yeasts in media COD? taining glucose and 5 mM KNO3 . . Production Of N 0 in late stationary phase by severaI fungi in media con- taining glucose and 5 mMLKNO3 . . Production OfN N0 in_ late stationary phase by severaIN 03 -reducing bacteria in synthetic media containing 5 mM KNO3 . . . . . . . Rates of NO production from NO2 by several migrobes . . . . . Production of N20 by plant t0ps. . Influence of chloramphenicol on NO production by diced Spinach leavego. man. 0 O O O 0 Levels of N20 in human breath before and_ after a meal containing NO3 and N02- 0 O O O O O O O Page 11 12 13 15 17 19 21 22 24 26 27 Table 12 13 Page 15N'MPN experiment in nutrient broth using Sloan loam, with 14 day incup bation at 25 C . . . . . 40 15N'MPN experiment in tryptic soy broth using SloaB loam, with 14 day incubation at 25 C . . . . 41 vi Figure LIST OF FIGURES Relation between N 0 production and phase Of growt in Serratia marcescens o o o o o 0 Relation between N20 production and phase of growth in Rhodotorula sp. Pattern of N 0 production from N02- by resting c3113 of Klebsiella pneumoniae taken in early stafionary phage. Krrows indicate points of NO2 addition. . . . . . NED content of breath before and a tgr ingesting a meal high in N03 and N02 0 Q 0 O O 0 vii Page 14 18 23 28 INTRODUCTION AND EXPERIMENTAL OBJECTIVES Both nitrate and nitrite can be converted to nitrogenous gases by chemical and biological processes. One such gas, N20, has received much attention, since it may act to deplete the Earth's ozone layer (12), and help promote an atmospheric greenhouse effect (40). Production of N20 from several chemical mechanisms involving N02- (27, 28, 34, 39, 45) or NHZOH (6) in laboratory experiments has been reported. The significance of these mechanisms in nature has not been demonstrated. It may be that most of the N20 produced in nature is due to biological processes. Of these, nitrification and denitrification have received the most attention. Although it has been known for some time that nitrifying bacteria can produce N20 (46), it has not been intensively investigated until recently (16, 20). Studies by Blackmer and Bremner (4) indicate that application of ammoniacal fertilizers to aerobic soils can result in significant losses of N20. Denitrifying bacteria are those which use nitrogenous oxides as electron acceptors to generate ATP under anaerobic conditions. In the process, the majority Of N03- or N02” is converted to N2 or N20 (29), with the prOportion of N20 produced being dependent on several environmental factors. Firestone §t_§l;’(l4) found that increases in nitrate, nitrite, oxygen and soil acidity cause N20 production to increase relative to N2. Studies with pure cultures of denitrifiers have shown NO in addition to N20 and N2 to be produced during denitrification (3), but the role of NO in denitrification is still controversial. Some bacteria can accomplish the dissimilatory 1 2 reduction Of N02- to NH4+ (10, ll, 33), apparently done to reoxidize reduced pyridine nucleotides during fermentation. Work by Yoshida and Alexander (46) and in our laboratory (37) led to the suspicion that these bacteria could produce N20 as well as NH4+, and that the presence of carbon could reduce N20 production. Yoshida and Alexander (46) and Bollag and Tung (5) had found that certain fungi could produce N20 from N02-. Work with green plants has indicated that nitrogenous oxides might be released from their foliage (35, 36, 41). Kaspar and Tiedje's finding that N03- and NO2- are dissimilated to NH4+ and N20 in the bovine rumen (22), coupled with the existence of similar organisms in the gastrointestinal tract, raised the question of whether N20 might be found in animal breath. This study was conducted as a survey of possible sources of N20 that had not received prior attention. Organisms reported to have the capacity to assimilate N03” into cell material (18, 29) were thought worthy of investigation. I report here on various microbes that produced N20 but not NO, as well as the production of N20 by damaged plant tops and in human breath. My research centered on the following questions: For pure cultures of microorganisms, 1. Which physiological groups of organisms are able to produce N20 from.NO3- or N02”? 2. At what stage of growth does N20 production occur? 3. What is the effect of carbon source on N20 production? 4. Is the production of N20 by these organisms regulated by ammonium? 5. Is NO produced by any of these organisms? 6. What are the rates of N20 production from N02“ by these organisms? For green plants, 1. Is there evidence to support the production of N20 by plant tissue? For human breath, 1. Is N20 found in human breath at levels exceeding the ambient concentration of the atmosphere? 2. Does the consumption of NO3'/N02' in the diet cause a change in the level of N20 in breath? MATERIALS AND METHODS Microorganisms The bacteria studied included Escherichia 991i.K12, Serratia marcescens, Enterobacter aerogenes, Klebsiella pneumoniae, Erwinia caratovora, Bacillus subtilis, and Acinetobacter sp. Clostridium.KDHS2 was isolated by W. H. Caskey (8). Vibrio succinqgenes was from the laboratory of Dr. C. A. Raddy. Dr. Harold Sadoff provided cultures of Azotobacter vinelandii strains A;.vinelandii 12837, A;_vinelandii nif-l2, A;_giggr landii rifr nif-64; and Azotobacter macrocytogenes strains A&_macrocytogenes 8700 and.A;_macrocytogenes 9129. The following fungi and yeasts were obtained from Dr. A. Rogers: Alternaria sp., Aspergillus sp., Fusarium sp., Helminthosporium.sp., Penicillium sp., Actinomucor ele ans, Candida tropicalis, Rhodotorula sp., and Hansenula sp. 13N-studies Pure cultures Of bacteria were grown aerobically in 500 ml Erlenmeyer flasks which contained 250 ml Of 5% tryptic soy broth (Difco) with 3.5 mM KN03. Cells were grown at 300 C on a rotary shaker at 150 rpm. After 12 h cells were harvested by centrifugation, washed in.0.05 M Tris buffer (pH 7.0), and resuspended. Cell suspensions of 0.5 ml were injected by syringe into serum vials containing 5% tryptic soy broth.without nitrate, under a helium headspace, with Ti(III)citrate to establish a lOW'Eho Autoclaved cells were prepared in a similar manner and served as a sterile control. TO initiate the experiment, 13N03-/13N02‘ (approx. 1 mCi) produced at the MSU cyclotron (38) and mixed with.unlabeled KNO3 was injected into each vial to achieve a nitrate concentration of 104nm. The vials were agitated on a rotary shaker for 20 min at 25° C after which the headspace gas was analyzed for 3N-gases by gas chromatography-proportional counting (38). Each vial was then Opened, and the medium.clarified by filtration through a 0.22 am filter. The medium was analyzed for 13N--ions by radio-HPLC (38). Conditions for assessment Of N30 production in batch culture Pure cultures of bacteria, yeasts and fungi were grown in 26 ml Balch tubes (Bellco Glass, Vineland, NJ), which contained 5 ml of the respective media. Media were amended with 5 mM.KN03, unless stated otherwise. The enteric bacteria and Bacillus were usually grown in 1.5% (w/v) tryptic soy broth. Potato dextrose broth (Difco) was used to culture all yeasts and fungi. Selected yeasts and fungi were also grown on a synthetic NH4+-free medium, prepared as follows. The following stocks (g/l) were prepared and autoclaved separately: Solution A-- KZHP04, 160.0; KH2P04, 40.0; NaCl, 10.0: Solution B—- MgSO4'7H20, 20.0: Solution C-- CaClZ-2H20, 2.5; FeCl3°6H20, 0.25. Stock vitamin and trace mineral solutions as described in (l) were prepared and sterilized separately. The synthetic medium.was prepared by adding 10 ml Of each stock solution to one liter of double- distilled water containinglKNO3 and glucose, and adjusting the pH to 5.1 with l N HCl. The Azotobacter strains were grown in Burk's medium (42), withKNO3 substituted for NH4N03. Acinetobacter was grown on a medium.of (g/l): Na acetate, 2.0; KN03, 2.0; and MgSO4'7H20, 0.2, prepared in 0.04 M KH2P04 and NazHPO4 buffer (pH 6.0). To this was added 1% (v/v) of the same trace mineral solution as above. Clostridium KDHS2 was grown on the medium of Caldwell and Bryant (7) except that soluble starch and cellobiose were omitted, and.KN03 was added. Vibrio was grown on the medium Of Wolin, Wolin and Jacobs (44). The fungi, yeasts, Azotobacter and Acinetobacter cultures were incubated under air. The enteric bacteria, Bacillus, yeasts and obligate anaerobes were incubated under 02-free argon, achieved by evacuating and flushing each tube three times. Tubes were inoculated with a 1-6% inoculum.from a seed culture. Tubes were positioned horizontally, and shaken at 100 rpm on a rotary shaker. Incubation was at 25° C in the dark, except for the enteric organisms, Bacillus and Vibrio which were incubated at 310 C. The fungi were grown as above, inoculated either via syringe from.sporulating slant cultures flooded with sterile saline, or by an inoculating loop scraped across such cultures. Culture tubes Of fungi were incubated stationary and vertical, except prior to gas analysis when they were shaken to ensure gaseous equilibrium. Resting cell studies Seed cultures of Escherichia ggli_Kl2, Klebsiella pneumoniae and Enterobacter aerogenes were grown in 40 ml Of tryptic soy broth without glucose, with 5 mM KNOB. Incubation was at 25° C with shaking at 100 rpm in Erlenmeyer sidearm flasks. Cultures were grown for 1 day (early stationary phase), then their entire contents were aseptically transferred to 450 m1 of the same medium in 500 m1 Erlenmeyer flasks. These flasks were capped with rubber stoppers pierced by one-way check valves (Nupro CO., Willoughby, OH), to relieve gas pressure. Incubation was stationary, at 250 C, for 1-2 days. Since Hansenula sp. grew best aerobically, its volume of medium was the same as above, but in one liter flasks. It was grown in potato dextrose broth with 5 mM.KN03. Stationary phase cultures were harvested by centrifugation at 10,000 X‘g for 10 min. Pelleted cells were resuspended in 10 m1 of the same medium without nitrate, plus 2003ug'ml-1 chloramphenicol. Cells were kept on ice for no more than 12 h until used. Cells were added to 40 ml of the initial growth medium without KN03, plus 200aug’ml'ml chloramphenicol, in presterilized 125 m1 Erlenmeyer flasks having Hungate sidearms. Each flask was connected to the recirculating gas assay system described by Kaspar and Tiedje (21). The system was modified to allow the semicontinuous sampling of gases from four flasks. Magnetic stirrers afforded continuous agitation of the cultures, and aided maintenance of equilibrium between gaseous and liquid phases. After making the flasks anaerobic by flushing with argon, 2 m1 of sterile 5 mM NaN02 was added to each culture. Gas samples were usually taken 7 every 20 min. At termination Of each experiment, cells were saved for protein analysis. Greengplants Since plants accomplish the light-driven reduction of N02' to NH4+ within their chloroplasts (13, 24, 26), plant tissue was investigated as a possible source of N20. For plant top analyses, seedlings in the field were uprooted, and their roots kept in water during transport to the laboratory. The seedlings were rinsed under tap water to remove as much soil adhering to foliage as possible, and blotted on paper toweling. Plant tOps were removed and placed into serum bottles, then sealed with butyl rubber septa and aluminum crimp caps. The headSpace of some bottles was air, termed aerobic. Others were evacuated and flushed three times with argon gas, and are termed anaerobic. Incubation was at 32° C. For the diced leaf experiments, fresh spinach was purchased at local markets and refrigerated until washing. Leaves selected for their wholeness and fresh appearance were rinsed under cold tap water to remove soil, then blotted on paper toweling. Leaves were then placed flat on plastic trays, covered with clear plastic wrap, and incubated in a growth chamber at 5-10° C under incandescent lights for 4-12 h, to revitalize their photosynthetic apparatus (9). Selected leaves were cut into approx. 1 cm? pieces. Leaves were large enough so that eight pieces could be cut from each. Four pieces of leaf were put into serum bottles, each bottle containing pieces from only one leaf. Ten milliliters Of 5 mMIKNO3 or NaNO2 was pipetted into each bottle; chloramphenicol, when included in this solution, was at 200.ug'ml'1. Bottles were sealed, then evacuated and flushed three times with argon to afford infiltration Of the nitrogen solutions into the leaf tissue; a slight modification of the method used by Klepper (23). Evidence of infiltration was taken as bubbles forming on the leaf surface under vacuum. After drawing the third vacuum, bottles were brought to atmospheric pressure, and reopened. After the aqueous phase was poured off, the bottles were rescaled under room air. Dark treat- ment bottles were covered with aluminum.foil. All bottles were incubated under incandescent lights at 35° C. Human breath The effect Of high nitrate/nitrite levels on N20 in breath was examined in five individuals by comparing N20 content of breath before and after eating. Samples of breath were Obtained by having subjects hold their breath for 15-20 sec, then exhaling into the plastic inlets Of one liter Saran bags (Markson Scientific Inc., Del Mar, CA) capped with rubber septa. Each person used a separate bag throughout the experiment. Bags were evacuated and flushed three times with argon between samplings to eliminate any N20 carryover. At two hours and one hour before eating, samples of each subject's breath were taken to provide individual background N20 values. These two values varied little for each person; so the two values were averaged and equated tO one. The data reported are the change in N20 at each post-meal sampling, referenced to the pre-meal mean for that individual. The five subjects ate a high N03-/N02- lunch of spinacheand-bacon salad. Fresh Spinach is reported to contain 69—541 ppm N03- on a fresh weight basis (25), 9 and bacon 20-50 ppm N02”. Each individual ate approximately 100 g of spinach. Analytical methods Except for the resting cell experiments, gas sampling was done by removing 0.25 ml gas samples with 0.5 ml glass syringes fitted with 25 gauge stainless steel needles (Becton, Dickinson and Co., Rutherford, NJ). Needle tips were capped with rubber stoppers to prevent leakage until samples were analyzed. Unless otherwise noted, injections were made onto a Perkin—Elmer Model 910 gas chromatograph, with Porapak Q columns at 50° C, and dual 63Ni electron capture detectors operated at 3000 C. Carrier gas was 5% CH4-95% Ar with a flow rate of 15 m1 min-1. Peak areas were determined with computing integrators. The lower level detection limit for N20 on this gas chromatograph was approx. 0.1 ng N/Ml gas; for N0, the lower level detection limit was approx. 1.0 ng N/Ml gas (21). , The Carle gas chromatograph used for some analyses had a Porapak 0 column at 30° C and a microthermister detector. Carrier gas was helium.with a flow rate of 15-19 m1 min-1. Its lower level detection limit for N20 was 560 ng N/ml gas. Presence Of N03'/N02- in cultures was determined by spot tests with diphenylamine reagent (30). Detection limits for this reagent were 100,uM N0 and lOMM N02”. Protein determination was by Lowry method (19). Growth of microbial cultures was monitored turbidimetrically with a spectrophotometer at 640 nm wavelength. 3 10 RESULTS Evidence that nitrate-respiring bacteria produced N20 is shown in Table l. The results in Table 2 show that the presence of glucose reduced the amount Of N20 produced by the Escherichia and Enterobacter species, and that neither N2 nor N0 were produced. Failure to detect N2 indicates thatBthese bacteria are not denitrifiers. Absence of N gas production by autoclaved cells confirms that the mechanism.was biological. Further evidence of the non-denitrifying nature of enteric organisms and Bacillus is provided by the work Of Smith (33). Whereas acetylene blocks the reduction Of N20 to N2 by denitrifying organisms (2, 47, 48), Smith (33) found no increase in N20 production by Citrobacter and Bacillus isolates in the presence of acetylene. Evidence for the dissimilation of N03“ to NH4+ by nitrate—respiring bacteria is shown in Table 3. Under these conditions N20 did not constitute more than 5% Of the 13N gaseous products (Table 2). Ammonium.was the major product (Table 3), and label associated with the cells was insignificant. Growth studies with two enteric bacteria showed that they produced N20, but only after reaching stationary phase. This is shown by the Serratia growth study (Figure l). Glucose slowed the rate of N20 production in Escherichia 921; (Table 4). With glucose, production of N20 after 2 days was slight- ly more than that after 5 h without glucose. The yeasts, like the enteric bacteria, produced N20 only in the stationary phase. Hansenula started producing N20 a few hours after growth ceased (Table 4). but its production was three orders of magnitude below that of the enteric bacteria. Generally, the enteric bacteria produced micromolar 11. Table 1. Production Of nitrous oxide by several bacteria.a Organism % N03'-N recovered as Nzo-Nb 12 h 2.5 days Escherichia coli 16 36 Klebsiella pneumoniae 11 30 Erwinia caratovora 19 Serratia marcescens 12 Enterobacter aerogenes 10 6 Bacillus subtilis 5 aGrown in 3% tryptic soy broth with 3.5 mM KN03. bAnalyses done with a Carle gas chromatograph with microthermister detector. 212 Table 2. Production Of 13M gases by enteric bacteria as influenced by glucose. Organism Glucose 13N20 13N2 + 13N0 (counts) (counts) Escherichia colj --- 25,581 0 V Escherichia cOli + 0 0 Escherichia coli, autoclaved --- 0 0 Enterobacter aerogenes --- 19,237 0 Enterobacter aerogenes + 263 0 13 Table 3. Reduction Of 13N03' and 13N02' to 13NH4+ by several bacteria.a Sample Ratio Of 13M ions found 13NH4+ 13N02’ 13N03' Source 0 20 80 Enterobacter aerogenes, autoclaved 0 22 78 Escherichia coli 100 18 82 Enterobacter aerogenes 100 0 0 Klebsiella pneumoniae 100 0 0 Bacillus subtilis 100 0, 0 Erwinia caratovora 100 0 0 aCultures incubated 20 min with 13N03- diluted with 10 UN unlabeled KN03. 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D. 1.0 aerobically and 0.92 anaerobically) under both conditions. It was also the only yeast that did not produce N20 (Table 5). Hansenula and Rhodotorula both achieved far higher cell densities when grown aerobically than anaerobically (0. D. 1.1 vs 0.49, and 0.93 vs 0.33, respectively), and both produced N20 (Table 5). The fungi seemed to have the weakest N20 generating ability of any group studied (Table 6). The initial amounts Of N20 assayed in these cultures did not increase much over time. When grown in potato dextrose broth, every N20- producing organism still had N03" or N02- left at termination of the assay. But when Hansenula, Aspergillus and Alternaria were grown in a NH4+-free synthetic medium, N03" and N02” were consumed complete- ly, and no N20 was formed. Only after Hansenula received additional N02- did N20 production start. 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The Acinetobacter (Table 7) and several fungi (Table 6) did not produce N20, either. The role of ammonium as a regulator Of N20 production is not obvious from these results. All the Azotobacter cultures had N03‘ or N02- remaining at the end of the experiment. But the three A;_vinelandii strains produced N20 in stationary phase (Table 7). while the A; macrocytogenes strains produced none. Rates of N20 production by resting cells of several of the N20 producing cultures are summarized in Table 8. After addition Of nitrite, most of the organisms exhibited a linear rate of N20 production, followed by a plateau region. The linear regions were used to estimate rates Of N20 production. When §;_pneumoniae was given a second nitrite addition, it exhibited another linear rise, then leveled Off again (Figure 3). This was interpreted to mean that all the N02“ had been dissimilated to NH4+ and N20 by the time a plateau was reached. The data confirmed that the organisms studied produced N20 but did not consume it. The rate studies were done in the recirculating system of the gas chromatograph, where 02-free conditions can be carefully maintained and monitored (21). This is necessary for a sensitive assay Of N0, since N0 quickly breaks down when it reacts with O2 (15). None of the organisms exhibited any measurable NO production. Plant tOps incubated in bottles Often produced N20 (Table 9). Most longbterm anaerobic incubations produced more N20 than short-term ones. Studies which included leaves treated with chloramphenicol 21 .omouapo .>\:. ww.o voc_eucouu .wrooaeo.-imoz aneuep.semme who: msm_:emao Lozuo _—< .Loccee zuouopasemmau a :. inoz ueuaemzo .euauooe oz A>\z. NN.o vecemucoue .mco_ue.>oe wheezeum Anemozucmhca cav.fl .mcems one mos—e>o .omou:_m .>\x. Ne.o eocaoucoun .sa. co" as :mzezm measupao Auoom an ueucaao:_ _. Louuoaouo~< oNo.o .~_v ma an m.x.=N :. emo.o Ao.a. mu Na emouapw we manage. p.5cepoc.> amuucnouo~< v~.o nae. mm a_ . s:_vms m.za:m :_ Nvo.o u.m.N. N_ Nu nomads—w N_ a.= v.5copoca> nouucnouo~< A». .mzce. zich ou ziinoz oNz omega aneco_ueum muaaom cszpcos we :O.mao>cou mo_os c ca «a.» coping use amazemho .85. z... m 2.53:8 2.6... Ovuezucxm :. e_amuoen acaoaeoa--moz pcae>mm an omega zya=o_ueum camp c. oNz No :O_uoanha .N open» 223 Table 8. Rates Of N20 production from N02‘ by several microbes. Organism Rate of N20 production (n mOl N20 min'1 mg protein' )a Escherichia coli K12 0.28 Klebsiella pneumoniae 0.14 Enterobacter aerogenes 0.11 Hansenula sp. 0.04 aProtein was measured by the method of Lowry (19), with bovine serum albumin as the standard. 23 8a .cowuecum -Noz 4o weapon mueueecw mzoha< .mmmza zgecoeusum apaem cw :mzep weecosamca oppmemampz 4o mppmu mc_umma an -Noz seam copuosuoaa ONz 4o campus; .m seamed mMHDZHE 8% cm 8— sfi We . so .9 .8 W N 0 .8. m .n. .93 S N .SNPACU .Sw niiiiiiiiav1 .eam 24' Table 9. Production of N20 by plant tOps. Speciesa Atmosphere Incubation period nmoles N20 (hours) in bottle Amaranthus retroflexus Air 8b 18 (Redroot pigweed) Capsella bursa-pastoris Air 6 23 (Shepherd's purse) Rumex sp. Argon 72 164 (Dock) Plantago sp. Argon 72 157 (Plantain) Stellaria media Argon 72 102 (Common chickweed) Acer negundo Argon 72 3 (Boxelder) Atmosphere Argon 1 aAll incubations at 32°C. Each bottle contained one plant tOp. bIncubated under incandescent lights. All others incubated in the dark. 25 seemed to bear out that microbes, not the plant tissue itself, were producing N20 (Table 10). In the diced leaf experiments, the illuminated samples produced less N20 than did the dark incubations. This may have been due to production of 02 by the chloroplasts in the illuminated samples, which could inhibit No3“ reduction. Results of the human breath experiment are shown in Table 11. Random spot testing Of peOple's breath had previously shown that some samples exceeded ambient atmospheric N20 levels. This experiment was designed to see if a meal high in N03'/N02- could raise the N20 levels of breath. A statistically significant increase in breath N20 content was noted after the meal. The most dramatic increase seen in a subject is shown in Figure 4. DISCUSSION The Bacillus and enteric bacteria that dis- similated nitrate to ammonium in tryptic soy broth were the most rapid and prolific producers of N20 from N03-. N20 production by Escherichia and Enterobacter was slowed in the presence of glucose. Such an effect makes sense if the glucose allowed fermentation to proceed to a greater extent, delaying the onset of stationary phase and N20 production. Smith (32) found this effect in tryptic soy broth, but not in nutrient broth. It would appear that the effect Of glucose upon N20 production by enteric bacteria can vary with nutrition. Every organism which produced N20 did so only after growth had ceased. In addition, the presence Of reduced forms of nitrogen did not seem to affect N20 production. The production of N20 did not seem 265 Table 10. Influence Of chloramphenicol on N20 production by diced spinach leaves in air. Incubation length Treatment 520 production (nmoles per bottle) (hours) Without With chloramphenicol chloramphenicola 9 Lightb o o Darkc 0 0 15 Light 32 (SM 0 Dark 158 (93.6) 0.002 (0.0006) 21 Light not done 0 Dark not done 2.2 (0.76) a200mg ml"1 chloramphenicol. Bottles in growth chamber at 32°C with two incandescent lights. cBottles covered with aluminum foil in the growth chamber. dValues are means, :_(in parentheses) standard deviations. 27' Table 11. Levels of N20 in human breath before and after a meal containing N03' and N02‘. Time (hours) N20 Level Of significance (%)a --Before eating-- -2 1 (0.06)b -1 1 (0.06) --After eating-- 1 1.30 (0.25) 90 2 1.37 (0.46) 80 3 1.32 (0.28)C 80 aEvaluated by two-tailed t test. bValues are means :_(in parentheses) standard deviations. cCalculated from four subjects; all other values from five subjects. 28 .-Nez eee -mez e. gee. Fame s mcwpmmmcw Lopes new whoemn gunman mo peopcoo oNz .5 shame; :3. N... :8 N8 N8 - A N.. a. a. r. . a. Im+ IN... Ia... IHI Iml lam—S. Kurt? m0 UKDLUD mmDDI 29 to be related to the assimilatory N03--reduction pathway, since this pathway is repressed by NH4+ (29). Instead, nitrous oxide could be produced by enzymes independent of any previously described. Smith (32) found that chloramphenicol prevented the induction of N20—producing activity in a Citrobacter soil isolate, indicating that it produces N20 enzymatically. He also found that three §;_ggli mutants lacking NADH-dependent dissimilatory nitrite reductase produced N20 at rates equal to the wild type, but released NH4+ at a much slower rate. Satoh 23.31, (31) isolated mutant strains of §;_pneumoniae that were defective in the reduction of N02- to NH4+, but which produced N20 at rates comparable to the wild type. These findings suggest that N20 is not a side product Of dissimilatory nitrite reduction to ammonium. ‘ Although the mechanism of N20 production is uncertain, the fact that it is produced only in stationary phase suggests that it may be produced by enzymes of secondary metabolism. Since most soil microorganisms grow very slowly, existing essentially in stationary phase, N20 production in nature by the microbes I examined seems reasonable. The level of detection for NO on the gas chromatograph should have allowed me to detect 2% conversion of N02'-N to NO-N. However, no strong evidence for NO was found. This indicates that free N0 is not involved in the pathway to N20 of these organisms; whereas N0 has been found under the same assay conditions for denitrification (3). If any of the plant materials had begun production Of N20 soon after incubation started, the role of the plant itself might have been more at issue. But production of N20 was never noticeable before 6 to 8 h, 30 most likely because it was not until then that microbial populations reached adequate levels to produce detectable amounts of N20 from.nitrate present in plant tissue. The role of microbes in producing N20 from.plants was best supported by the chloramphenicol experiment, in which inclusion of chloramphenicol with the nitrite solution prevented significant N20 production. The plant tissue examined was damaged. The act of pushing the plant tOps through the narrow mouths of serum bottles almost always resulted in visible bruising or laceration of the plant tissue. In the diced leaf experiments the tissue was exposed to a vacuum, also causing damage. Damaged plant tissue may be the rule in nature rather than the exception, due to insects, winds and other influences. Making a cautious extrapolation to field situations, it may be that microbes growing upon damaged plants can account for some N20 production. In a recent study (17), 167 strains Of Serratia were isolated from.623 plant samples. If such bacteria were to colonize damaged plant tissue rich in nitrates, the release of N20 from within anaerobic Sites in plant tissue might ensue. Might plant tissue ever produce N20 by itself? Using the microbial studies as a model, perhaps the ability exists in some plant tissues at a physiological stage corresponding to stationary phase in microbes. Autumn might be the best time to look for such activity, in senescing plant tissues. The analysis of breath samples showed that the level of N20 in human breath can rise significantly above that of the atmosphere. The point in the human body from.which N20 originates is problematic. Microbial flora in the gut or oral cavity could be 31 two possible sources; different diets and levels of dental hygiene may account for differences in N20 levels in the breath of different people. Perhaps the purported nitrifying activity Of human tissue itself (43) can lead to release of N20. This study found that N20 can be released from microbial, plant and.animal sources. It is possible that microbes are the true producers in each case. The flux of N20 from these sources may significantly contribute to the N20 flux into the Earth's atmosphere. Although the percentages of N20 these sources produce are small, the extent of these sources is large. l. 3. 4. 5. 7. 9. LITERATURE CITED The American Type Culture Collection: Catalogue of Strains I. 1978, 12th ed. American Type Culture Collection, Rockville, MD. Page 354. 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. Betlach, M. R. 1981. A kinetic explanation for accumulation of nitrite, nitric oxide, and nitrous oxide during bacterial denitrification. Appl. Environ. Microbiol. 42:1074-1084. Blackmer, A. M., J. M. Bremner, and E. L. Schmidt. 1980. Production Of nitrous oxide by ammoniae oxidizing chemoautotrophic microorganisms in soil. Appl. Environ. Microbiol. 40:1060—1066. Bollag, J. M. and G. Tung. 1972. Nitrous oxide release by soil fungi. Soil Biol. Biochem. 4: 271-276. Bremner, J. M., A. M. Blackmer, and S. A. Waring. 1980. Formation Of nitrous oxide and dinitrogen by chemical decomposition of hydroxylamine in soils. Soil Biol. Biochem. 12:263-269. Caldwell, D. R. and M. P. Bryant. 1966. Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Appl. Microbiol. 1437 94-801 0 Caskey, W. H. and J. M. Tiedje. 1979. Evidence for Clostridia as agents of dissimilatory reduction Of nitrate to ammonium in soils. Soil Sci. Soc. Amer. Jo 43:931-936. Clark, J. M. Jr. and R. L. Switzer. 1977. Experimental Biochemistry, 2nd ed. W. H. Freeman and Co., San Francisco, CA. Pages 293-294. 32 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 33 Cole, J. A. 1978. The rapid accumulation of large quantities of ammonia during nitrite reduction by Escherichia coli. FEMS Micro. Letters 4:327—329. Cole, J. A. and C. M. Brown. 1980. Nitrite reduction to ammonia by fermentative bacteria: a short circuit in the biological nitrogen cycle. FEMS Micro. Letters 7:65-72. Council for Agricultural Science and Technology. 1976. Effect of increased nitrogen fixation on stratOSpheric ozone. Report NO. 53. Iowa State University, Ames, IA. Dalling, M. J., N. E. Tolbert, and R. H. Hageman. 1972. Intracellular location of nitrate reductase and nitrite reductase. Biochim. BiOphys. Acta 283:505—512. Firestone, M. K., R. B. Firestone, and J. M. Tiedje. 1980. Nitrous oxide from soil dani- trification: factors controlling its biological production. Science 208:749-751. Gerber, E. A. E. and T. C. Hollocher. 1981. 15Ne tracer studies on the role of N0 in denitrification. J. Biol. Chem. In press. Goreau, T. J., W. A. Kaplan, S. C. Wofsy, M. B. McElroy, F. W. Valois, and S. W. Watson. 1980. Production of N0 and N 0 by nitrifying bacteria at reduced concefitrationg of oxygen. Appl. Environ. Microbiol. 40:528-532. Grimont, P. A. D., F. Grimont, and M. P. Starr. 1981. Serratia species isolated from.plants. Current MicrOSiol. 5:317—322. Hall, J. B. 1978. Nitrate-reducing bacteria, in D. Schlessinger, ed. ‘Microbiologyh-l978. Kierican Society for’MicrObiology. Hanson, R. S. and J. A. Phillips. 1981. Chemical composition, igDP. Gerhardt, ed. Manual of . Methods for General Bacteriology. American Society for'MicrobiOlogy. Pages 358-359. Hooper, A. B. and K. R. Terry. 1979. Hydroxyl- amine oxidoreductase of Nitrosomonas: production of nitric oxide from.hydroxylamdne. Biochim. Biophys. Acta 571:12-20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 34 Kaspar, H. F. and J. M. Tiedje. 1980. Response Of electron-capture detector to hydrogen, oxygen, nitrogen, carbon dioxide, nitric oxide and nitrous oxide. J. Chromatog. 193:142-147. Kaspar, H. F. and J. M. Tiedje. 1981. Dis- similatory reduction of nitrate and nitrite in the bovine rumen: nitrous oxide production and effect of acetylene. Appl. Environ. Microbiol. 41:705-709. Klepper, L. A. 1975. Inhibition of nitrite reduction by photosynthetic inhibitors. Weed Sci. 23:188-190. Magalhaes, A. C., C. A. Neyra, and R. H. Hageman. 1974. Nitrite assimilation and amino nitrogen synthesis in isolated spinach chlorOplasts. Plant Phys. 53:411-415. Maynard, D. N., A. v. Barker, P. L. Minotti, and N. H. Peck. 1976. Nitrate accumulation in vegetables. Advances in Agron. 28:71-118. Miflin, B. J. 1974. Nitrite reduction in leaves; studies on isolated chloroplasts. Planta (Berl.) 116 3 187-196 0 Nelson, D. W. and J. M. Bremner. 1970. Role of soil minerals and metallic cations in nitrite decomposition and chemodenitrification in soils. Soil Biol. Biochem. 2:1-8. Nelson, D. W. and J. M. Bremner. 1970. Gaseous products Of nitrite decomposition in soils. Soil Biol. Biochem. 2:203—215. Payne, W. J. 1973. Reduction of nitrogenous oxides by microorganisms. Bact. Rev. 37:409-452. Rowe, R., R. Todd, and J. Waide. 1977. Micro- technique for most-probable-number analysis. Appl. Environ. Microbiol. 33:675-680. Satoh, T., S. S. M. Ham; and K. T. Shanmugam. 1981. Production Of nitrous oxide as a product of nitrite metabolism by enteric bacteria, 22 J. M. Lyons 33 al., eds. Genetic engineering of symbiotic niITOgen fixation and conservation of fixed nitrogen. Plenum Press, New York. Pages 481-497 0 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 35 Smith,o S. MM, 1981. Dissimilatory reduction of N0 by a soil Citrobacter sp. Appl. En iron. NH4Microbiol. 15 press. Smith, S. M. and K. Zimmerman. 1981. Nitrous oxide production by non-denitrifying soil nitrate reducers. Soil Sci. Soc. Amer. J. In press. Stevenson, F. J., R. M. Harrison, R. Wetselaar, 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 acidic conditions. Soil Sci. Soc. Amer. Proc. 34:430-435. Stutte, C. A. and R. T. Weiland. 1978. Gaseous nitrogen loss and tranSpiration of several crop and weed species. Crop Sci. 18: 887-889. Stutte, c. A., R. T. Welland, and A. R. Blem. 1979. Gaseous nitrogen loss from.soybean foliage. Agron. J. 71:95-97. Tiedje, J. M. 1981. Enhancing biological production of ammonia from.atmOSpheric nitrogen and soil nitrate, in J. M. Lyons et al., eds. Genetic engineering _Of symbiotic nitrogen fixation and conservation of fixed nitrogen. Plenum Press, New York. Pages 481-492. Tiedje, J. M., R. B. Firestone, M. K. Firestone, M. R. Betlach; M. S. Smith, and W. H. Caskey. 1979. Methods for the production and use of Nitrogen-l3 in studies of denitrification. Soil Sci. Soc. Amer. J. 43:709-716. Van Cleemput, 0., W. H. Patrick, and R. 0. Mc- Ilhenny. 1976. Nitrite decomposition in flooded soil under different pH and redox potential conditions. Soil Sci. Soc. Amer. J. 40:55-60. Wang, W. C. and Sze, N. D. 1980. Coupled effects of atmos heric N 0 and 03 on the Earth's climate. Nature 2 6:589-590. Welland, R. T. and C. A. Stutte. 1980. Concom— itant determination of foliar nitrogen loss, net carbon dioxide uptake, and transpiration. Plant Phys. 65: 403-406. 42. 43. 44. 45. 46. 47. 48. 36 Wilson, P. W. and S. G. Knight. 1952. Exe periments in Bacterial Physiology. Burgess Publishing Co., Minneapolis, MN. Witter, J. P., S. J. Gatley, and E. Balish. 1981. Evaluation Of nitrate synthesis by intestinal microorganisms in vivo. Science 213: 449-450. Wolin, M. J., E. A. Wolin, and N. J. Jacobs. 1961. Cytochrome-producing anaerobic vibrio, Vibrio succinogenes, Sp. N. J. Bacteriol. 81: 911- 917 e enzymatic formation Of nitrogen gas. Nature 210: 1150-1151. Yoshida, T. and M. Alexander. 1970. Nitrous oxide formation by Nitrosomonas eurogaea and heterotrOphic microorganISms. $01 01. Soc. Amer. Proc. 34:880-882. Yoshinari, T. and R. Knowles. 1976. Acetylene inhibition Of nitrous oxide reduction by denitrify- ing 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. APPENDIX APPENDIX FATE OF 15NO3 IN TWO COMPLEX MEDIA USED FOR MPN ESTIMATES OF SOIL DENITRIFIER POPULATIONS The most-probable—number (MPN) method allows estimation of microbial populations by the use Of dilution series. Given the highest dilution at which positive results are seen, statistical tables allow one to say what the most probable number of organisms in the inoculum was (1). I participated in an effort undertaken by the laboratory to validate the best method Of providing an estimate Of soil denitrifier pOpulations by the MPN procedure. The procedure developed is as follows: 10 g of refrigerated or fresh soil was placed in a blender containing 90 ml of 0.85% sterile saline solution plus one drOp of Tween 80. The suSpension was blended for 2 min, poured into a dilution bottle with a rubber stopper, and shaken to ensure suspension of the soil. One milliliter was withdrawn by syringe and injected into 9 ml of sterile saline solution. This tube was labeled the 10"2 dilution. One milliliter Of its suspension was removed and injected into 9 m1 of sterile saline solution, designated the 10"3 dilution. This procedure was continued until the 10'6 dilution was reached, so that five tubes containing dilutions of soil in saline solution were prepared. These were used to inoculate a five tube dilution series. One tenth milliliter was withdrawn from each saline dilution and used to inoculate 10 m1 Of sterile medium. The 10'2 saline 37 38 dilution was used to inoculate the five 10'3 culture tubes, and so on. The media was contained in Hungate tubes, a1- lowing injection of sterile acetylene into the tubes before inoculation with soil. Acetylene inhibits nitrous oxide reduction by denitrifying bacteria (2, 6, 7), and is used to confirm that the N03- is removed by denitrification. Tubes were incubated in stationary position at 20—30° C for 14 days. Tubes were then assayed for N20 production with a Carle gas chromatograph having a Porapak 0 column at 30° C and a microthermister detector. The carrier gas was helium with a flow rate of 15-19 m1 min‘l. Later, the medium in the tubes was assayed with diphenylamine reagent (4) for the presence Of N03—/N02-. After many trials, it was found that two media, tryptic soy broth and nutrient broth, afforded high estimates of soil denitrifier populations. However, when the two media were inoculated with the same soil, there would Often be an order of magnitude difference between the populations estimated by each. The fate of nitrate in the two media appeared to be different. Usually if 20% or more Of the NO3"-N in a nutrient broth tube was recovered as N20—N, all the N03-/N02' was gone. However, in tubes of tryptic soy broth, although NO3"/N02"' would be totally consumed in a tube, the amount of N03--N converted to N20-N could vary widely, but was usually well below 20% of the N03-N. To see if the two media did indeed promote two different fates of nitrate, an experiment using 15N as a tracer was designed. The MPN procedure was conducted as previously described, except that KlsNO3 was used. The soil used as an inoculum was a Sloan loam (Fluventic Haplaquoll). 39 After 14 days incubation, the tubes were analyzed for N20 production by gas chromatography. After steam distillation, cultures were assayed for the total amount of NH4+ present by Solorzano method (5). Ratio mass spectroscopy was then performed as described in (3), to find the amount of 15N03- that was converted to 15NH +. The results are shown in Table 12 and Table 13. Cultures in nutrient broth (Table 12) consistently produced a considerable amount of N20, with little ammonium.being formed from.nitrate as a rule. Tryptic soy broth, on the other hand (Table 13), usually fostered the production of large amounts of ammonium. but little N20, from.nitrate. The correlation between 20% or more of N03.-N converted to N20-N, and the disappearance Of N03-/N02' was very good in nutrient broth (Table 12), making nutrient broth the medium of choice for enumerating soil denitrifier populations. The correlation between appearance of N20 and disappearance of N0 -/N°2- was not good in tryptic soy broth (Table 13 . The large amounts of ammonium produced from.nitrate in these tubes indicated that this medium selected for dissimilatory ammonium? producing bacteria instead Of denitrifiers. Each medium.may prove useful to enumerate a different pOpulation in soil; nutrient broth to enumerate denitrifiers, and tryptic soy broth to enumerate bacteria that dissimilate nitrate to ammonium. 40 .NNNN—ece macaw; cozoam we: mazes .Ae. Deemed. m:_smpxcozame Na cezashouoao .Am. oce~no_Cm No venues x: cocashoumoa .saaome PE oz hm>o rosewood; NE 0 o» emote ace—zuooe NE .o 'li‘l'l-li. - e N.o e e N - o N.o o e e - e e.o a e N - ..e e.e e ..e N - ..o e.e o ..o . N-e. - N.e e.. N.N N.N N - ..e e.e ..e e e - e.e e.o e.e N - e.N e.. N.o N.. N + e.eN - e- e.eN . e-o. - N.e e.. N.N e.o m . N.NN ..N N.N e.NN e + N.eN N.N ..N. ..Ne N + e.NN e.N N.e ...N N . e.NN e.N o.oN e.Ne . N-e. + N.Nm ..N N.NN e.Ne N + N.NN N.. N.N N.eN e . N.NN e.. ..e N.eN N . N.NN e._ N.N e.NN N + e.NN e. ..m N.NN N e-e. . N.NN N.. a... N.eN N . e.eN NN. N.N e.NN e . N.em ... a... N.eN N . N.NN e. N.N ..NN . N + N..N N. N.N e.eN . N-eN cNNoor N no .ezz + eNz. z-e=zm. ea z-eNz ea -Nez heNeee -Nez as a. -Nez z-Nez mo z--Nez Le .+. museums no .u. monumoaa No zgo>oumn a aea:u\ziezz as :owmnemheu n copmco>coo a was» cONHSPNo eoeo.NNooe .e N . Nezx as N + reach Leo_cesz i .uomN we coaucaauca New eN sp.: .smo— coo—m chm: guano ace—nus: ca acmeaaoaxo zaz sz .NN mane» 41 .Ns. Ncouema mcaschcmzaac as cocNELouoco .Nm. ocm~aopom no assume as cocNELeumoa .s=_cos .5 ON am>o commence; N: o o» emote ocoquoom Na Na . o e.o o o m . N.o e.o N.o o c i o v.o o o N . N.o v.o o N.o N . N.o v.0 N.o o N NioN + m.cm N.NN v.mo N.m m . N.Nm N.NN N.NN ¢.NN e + o.em «.mN e.NN c.o m + N.Ne N.N o.Ne N.N N + N.Nm «.9 o N.NN N oioN + m.me N.N o.Ne N.N m + ¢.Nm v.w o.vm N.N e + c.NN m.v N.mN N.N m + N.NN o.m N.Nm m.mN N + N.Nm o.o m.m¢ N.N N m-oN + N.NN N.N N.NN N.o m + o.oa o.o N.mm s.m v + v.Nm N.NN N.NN o.N N + N.Nm N.o m.mm N.m N + e.we a.e n.mo N.N N eioN + o.mm N.e N.NN N.m m + o.NN N.e N.NN w.m e + N.NN o.v N.NN o.m . m + m.oo N.m N.Nm o.m N + N.NN o.m N.NN N.m N NioN canoe: N a. .3... 4 3.. 2-2.sz 3 5%.. 3 Ne: has... .82 do he -82 2-82 N do 238.. do N+N renames no N-N monumoaa No zgo>ooma s amaauxziczz as :eNNim :ou a co.mhm>:ou a was» soaps—No coco—Nudes Ne N + mozz :5 m + emoo=_m N>\3N NmN.o + zuoaa New oauazu» .uomN an =o_ucn=oc— Nee eN zNN: .ENoN coo—m chN: sworn Nam uwuaxgu cN aces—amaxo zaz sz .mN oNaep l. 2. 3. 4. 5. 7. LITERATURE C ITED Alexander, M. 1965. MOst-probable-number method for microbial pOpulations, in C. A. Black 31 al., eds. Methods Of SOiI_Analysis, Part 2--CEEmical and Microbiological Properties. Agronomy 9:1467-1472. Balderston, W. L., B. Sherr, and W. J. Payne. 1976. Blockage by acetylene of nitrous oxide reduction in Pseudomonas erfectomarinus. Appl. Environ. MicrObiOl. 31:50E-558. Kaspar, H. F. and J. M. Tiedje. 1981. Die- similatory reduction of nitrate and nitrite in the bovine rumen: nitrous oxide production and effect of acetylene. Appl. Environ. Microbiol. 41:705-709. . Rowe, R., R. Todd, and J. Waide. 1977. Micro- technique for most-probable-number analysis. Appl. Environ. Microbiol. 33:675-680. Solorzano, L. 1969. Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol. Oceanogr. 14:799-801. Yoshinari, T. and R. Knowles. 1976. Acetylene inhibition of nitrous oxide reduction by denitrify- ing 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. _42 lllllll iii 1627 my" Rfl 1 (with l 31 llllllllll