m mm m l m mu: 1m 11“! Linus” min; w; tum; u 3 1293 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records I ‘ . ‘ warm: m1 UHVFRSH‘! UTILIZATION AND PRODUCTION OF N20 BY DENITRIFIERS ISOLATED FROM DIFFERENT SOIL ENVIRONMENTS AND EFFECT OF pH ON THE RATES AND PRODUCTS OF DENITRIFICATION By Gilbert Uwahamaka Okereke A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crap and Soil Sciences 1978 ABSTRACT UTILIZATION AND PRODUCTION OF N20 BY DENITRIFIERS ISOLATED FROM DIFFERENT SOIL ENVIRONMENTS AND EFFECT OF pH ON THE RATES AND PRODUCTS OF DENITRIFICATION By Gilbert Uwahamaka Okereke A total of 88 strains which grew by denitrification in tryptic soy broth (TSB) were examined for their ability to grow on N20 as their electron acceptor as well as for their tendancy to produce N20 from NOB- in the absence and presence of acetylene. Eight strains did not grow with oxygen as the electron acceptor and three more did not grow with N03-. Thus 77 were confirmed as active denitrifiers for the survey. Sixty-four or 83% of the 77 strains reduced N 0, while 13 2 strains produced but could not use N20. One strain, 204, exhibited reduction of N03- to N2 but could not produce or use N20. Strains [Nos. 42, 44, 69, 110, 151] reduced N03- to N2 but apparently did not have the capacity to grow on N20. For most taxonomic groups 2/3 or more of the strains reduced N20. ‘However, none of the strains which clustered as Pseudomonas aeruginosa grew on N20. All strains of £4. stutzeri studied utilized N20 as a terminal electron acceptor. No strain of Pseudomonas sp. type 2 utilized N20. A high prOportion of E; fluorescens biotype II reduced N20. This was also the most commonly encountered denitrifier in the world survey of new isolates by Gamble, suggesting that the capacity for N20 reduction commonly exists in soils. The accumulation of N20 from N03- in the presence of acetylene by all but one of the isolates provides strong evidence that N20 is Gilbert Uwahamaka Okereke generally an intermediate in denitrification as well as provides additional support for the usefulness of this chemical as a general inhibitor of N20 reduction. Tryptic soy broth was found to be superior to nutrient broth as the medium base for denitrifier growth. Cell yield was linearly related to concentration of N20 (0.1 to 1 atm) in the incubation vessel for the four strains tested; thus high concentrations of N20 are not toxic. Cellular growth yields on N20 in batch culture ranged from 5.6 g cells/e- transferred for the fastest growing strain tested to 2.2 for slower growing strains. N20 when used in most probable number tubes as the only electron acceptor was not consumed at dilutions down to 10-3 per gram. Additions of fresh carbon source and N03- after growth did not stimulate N20 reduction. Since pure cultures of denitrifiers grew well under the same conditions this result was unexpected; the explana- tion has not been found. Limited studies were done on phase II denitrification rates in soils of different pH using the acetylene inhibition method in an anaerobic assay. Two of three very acid (pH 4 to 5) Nigerian soils showed significant denitrification in natural but not in autoclaved samples. This indicates presence of denitrifying enzymes in these soils. In contrast, four Michigan soils which had been decreasing in pH due to addition of different N fertilizer carriers showed little denitrification activity. The same soils which had recently been limed showed greater activity. The high activity in the pH 4.4 and 4.5 Nigerian samples suggest that acid tolerant denitrifying populations may have developed in these soils which had been acid for a very long period. The Michigan soils had become acid only recently. \ To my son Chinaedu and my father Ukaobasi. ii ACKNOWLEDGEMENTS I am very grateful to Dr. J. M. Tiedje, Chairman of my guidance committee, for this opportunity and for his support, understanding, guidance and encouragement throughout the duration of this study. I have to emphasize that I was very lucky to work in a laboratory 'where fellow graduate students were very friendly and always ready to help each other. Mary Firestone, Mike Betlach and Scott Smith are due special thanks for sharing their knowledge and experiences with me. I would also like to extend a warm thanks to my wife Victoria Ifeoma Okereke, my daughter Jenete Okereke and my little son Chinaedu Echezona Okereke for their patience, endurance and understanding. I thank the members of my guidance committee; Drs. B. G. Ellis and F. Dazzo for their encouragement. iii TABLE OF CONTENTS Page LIST OF TABLES O O O 0 O O O O O O O O O O O O O C O O O 0 O 0 v LIST OF FIGURES O O O I O O O O O O O O O O O O O O O O O O 0 Vi CHAPTER I INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O 2 UI MATERIALS AND METHODS O O O O O O O O O O O O I O I I O O 0 Description of denitrifer strains . . . . . . . . . . . Comparison of growth media . . . . . . . . . . . . . . Preparation of inocula . . . . . . . . . . . . . . . . Experimental culture conditions . . . . . . . . . . . . Analyses . . . . . . . . . . . . . . . . . . . . . . Growth yield experiments . . . . . . . . . . . . . . . Use of N20 as electron acceptor in MP tubes . . . . . \oooooxncxoxu: RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . 11 Incubation conditions . . . . . . . . . . . . . . . . . 11 Distribution of N 0 production and utilization capabilities among denitrifier strains . . . . . . . . 16 Growth Yields 0 O O O C O O O O O I O C O O O O O I O I 29 Use of N20 as an electron acceptor in MPN tubes . . . . 30 L ITERATURE CITED 0 O O O O O O O O O O O O O O O O O O O 0 35 CHAPTER II INTRODUCTION 0 O O C O O O O C O O C O O O O C O O C O O O O 39 SUMMARY OF PREVIOUS INVESTIGATIONS . . . . . . . . . . . . . 41 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . 43 $0113 0 C O C O I C C O C O O O O O O O O O O O O O O O 43 Assay of denitrification . . . . . . . . . . . . . . . 45 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . 47 Evaluation of sterilization methods . . . . . . . . . . 47 Denitrification rates in soils of different pH . . . . 47 Future work Q C C O O O O O C C C C O I O O O O O O C O 51 LITERATURE CITED 0 O O O O O O O O O O O O O O O O O O O O 0 53 APPENDIX A O O O O O O O O O O O I O O O O O O O O O O O O O 5 5 iv 10. 11. 12. 13. 14. LIST OF TABLES Characteristics of soils used . . . . . . . . . . . Comparison of complex media for supporting growth of denitrifier strains . . . . . . . . . . . . . . . . Comparison of rate of growth of P; perfectomarinus, §;_stutzeri and A; faecalis in nutrient broth and tryptic soy broth (TSB) with N20 as the only terminal electron acceptor . . . . . . . . . . . . Validation of Hungate tubes and He flushing regime for assaying N20 dependent growth . . . . . . . . Utilization and production of N20 by 79 denitrifier isolates and 9 reference cultures . . . . . . . . Number of denitrifier strains showing the indicated denitrification pattern . . . . . . . . . . . . . . Distribution of the capacity to grown N 0 among the similarity clusters of denitrifer strains found by Gamble SE El. (12) O O O O C O O O O O O O O O O C Major species that utilized N20 . . . . . . . . . Growth yields of several denitrifer strains when grown in batch culture on N20 as electron acceptor Characteristics of Nigerian soils used in study . . Characteristics of Michigan soils used in study . . Effect of autoclaving and addition of propylene oxide on pH of Saginaw soil . . . . . . . . . . . . Rate of denitrification in Nigerian soils of various pHS C O O O C C O O C O O I I O O O O O O O O O C 0 Rate of denitrification in Michigan soils; soils are paired to compare unlimed and lime treatment . . . Page 10 12 14 15 19 23 27 28 31 44 46 48 49 50 Figure 1. LIST OF FIGURES Page Growth of denitrifiers in different N 0 2 concentrations . . . . . . . . . . 17 Growth yield of four denitrifer strains with N20 as terminal electron acceptor 32 vi CHAPTER I UTILIZATION AND PRODUCTION OF N 0 BY DENITRIFIERS ISOLATED FROM DIFFERENT SOIE ENVIRONMENTS INTRODUCTION Nitrogen and nitrous oxide are considered the major products of denitrification (2, 13, 17). Whether nitrous oxide is produced and/or utilized by most denitrifiers is however a question to which no defi- nite answer has been given. This subject has been of considerable debate ever since nitrous oxide was recognized by Gayon and Duppet (14, 15, 16) as a gaseous product of the denitrification reaction. Several hypothetical pathways have been proposed, some treat nitrous oxide as an obligatory intermediate and others do not. The fact that nitrous oxide is one of the end products of deni- trification makes it extremely probable that this process is the source of this gas in the Earth's atmosphere. It was in 1911 that scientists predicted nitrous oxide as an atmospheric constituent of microbial origin and in 1938 its presence was verified by direct observation. In the early twentieth century there were conflicting ideas about the role of nitrous oxide in denitrification, partly because of inadequate techniques. For example Beijerinck and Minkman (3) and Suzuke (29) maintained that nitrous oxide was always present as the gaseous products of denitrification, whereas Gayon and Dupetit (16) and others claimed that nitrous oxide was entirely absent in some of their experiments. Sacks and Barker (26) rejected entirely nitrous oxide as an intermediate in nitrogen formation while Kluyver and Verhoeven (20) considered that nitrogen may have a dual origin: partly derived from a hydrogenation of nitrous oxide and partly from direct hydrogenation of the precusors of nitrous oxide. Recently greater interest in this topic has been stimulated by the hypothesis that nitrous oxide released to the atmosphere leads to the partial distruction of the ozone layer which protects the earth from biologically harmful ultraviolet radiation (6, 7, 8, 23). It has also been recognized that the use of nitrogenous fertilizers and other agricultural practices might increase the atmospheric concentration of nitrous oxide and thereby pose more danger to lives on Earth. Recent calculations by Wang 35 31. (30) show that if the nitrous oxide in the atmosphere is doubled, it would cause a warming of the planet that could drastically change the climate and thus be harmful to food production. These recent concerns about nitrous oxide and its hazardous effect to man and food production made this ignored product of deni- trification a topic of great interest. At the moment there are many basic questions yet to be answered concerning nitrous oxide production and utilization by denitrifiers. It is known that nitrous oxide is a trace component of the atmosphere and a major sink for nitrous oxide was considered to be photochemical dissociation in the troposphere and stratosphere (2); this has been supported by Schutz 35 a1. (27). Evidence that soil can also act as a sink for atmospheric nitrous oxide under certain conditions was obtained from studies showing (4) that soil microorganisms have the capacity to remove nitrous oxide from soil atmosphere until the concentration of this gas is much lower than the concentration in air. This uptake of nitrous oxide by soils was found to be due to microbial reduction stimulated by readily available organic matter. Certain denitrifiers can grow on nitrous oxide as the sole oxidant (9). Kluyver and Verhoeven (20) concluded that nitrous oxide is an intermediate in denitrification in at least some bacterial species because of the ubiquity of its occurrence. It has also been reported that Pseudomonas stutzeri (l), g; denitrificans (22) and Paracoccus (formerly Micrococcus) denitrificans (25) grew anaerobically using nitrous oxide as an electron acceptor. Although there exists some literature on nitrous oxide utilization and production by some denitrifiers, there has not been an extensive study of the nitrous oxide utilization and production by a wide variety of isolates from nature. To my knowledge, studies so far carried out have been on one or a few denitrifiers. Because of this, there are still differences in opinion among investigators concerning whether nitrous oxide can be utilized and produced by all denitrifiers and whether it is an obligatory intermediate in denitrification. As far as ecological interpretations are concerned, it is useful to identify denitrifiers of ecological importance for use in the study of the biochemical and physiological features of the pathway of deni- trification. In this regard work with pure cultures is important though care has to be taken when using the results to predict what happens in Nature. The purpose of this study was to survey the isolates of Gamble (11) for their ability to utilize and produce nitrous oxide. Other studies were undertaken to determine cell growth yields when grown with nitrous oxide as the terminal electron acceptor and to investigate (whether nitrous oxide as the only electron acceptor in MPN tubes could serve as a specific method for enumeration of denitrifiers. The results of these studies may help to elucidate the denitrification pathway. For instance organisms that reduce nitrate to nitrogen but cannot grow on N20 may help in elucidating the importance of nitrous oxide in respiratory nitrate reduction. Furthermore if nitrous oxide is the only gas produced from nitrate by growing cells of these organisms, then the study of the pathway of denitrification will not be complicated by the production of two gases as often occurs with other denitrifying organisms (20). MATERIALS AND METHODS A Description of denitrifier strains A total of 114 isolates confirmed as denitrifiers by Gamble (11) and 10 reference strains of denitrifying bacteria were studied. The reference strains were Pseudomonas fluorescens (ATCC 17822), Pseudomonas perfectomarinus, Hyphomicrobium sp. (WC 24 R, from Peter Hirsch), Pseudomonas denitrificans (ATCC 13867), Paracoccus denitrificans (ATCC 2008), Pseudomonas stutzeri (ATCC 17588), Pseudomonas aureofaciens (ATCC 13985), Pseudomonas mendocino (ATCC 25411), Pseudomonas aeruginosa, Alcaligenes faecalis (ATCC 8750). The origin of these strains is given by Gamble (11). Other strains used as controls to check for 02 con- tamination were Pseudomonas strains 388 and 402 obtained from G. E. Becker, University of Iowa and a Pseudomonas strain that grew on NTA. Becker strains 388 and 402 do not grow on N 0 while the NTA consuming 2 strain is an obligate aerobe. The 114 isolates were confirmed by Gamble to be denitrifers by the production of N20 and/or N2 during growth in nitrate broth Gamble (11). A list of these denitrifiers is found in Appendix A. These cultures were isolated by Gamble from soils, fresh water lake sediments and nitrified poultry manure and came from eight countries and a variety of different soils and environments. Comparison ofjgrowth media The stock cultures used were prepared by T. N. Gamble in sterilized soil in sealed screw cap tubes and had been stored two years in the refrigerator. Aggregrates of soil were aseptically transferred to test tubes containing 10 ml of sterilized nutrient broth (Difco) and tryptic soy broth (Difco). The cultures were incubated aerobically at 30° C. When turbid a loop of the culture was transferred to tubes containing 10 ml of each of the following three test media: (1) 0.8% nutrient . broth (Difco, Detroit, MI), (ii) 3% tryptic soy broth (TSB, Difco) and (iii) 3% TSB plus 3.5 mM KN03. The first two were incubated aero— bically at 30° C and the latter anaerobically in a glove box at room temperature. Growth was scored as visible turbidity after 7 to 14 days. The above complex media contain grams/litre: Tryptic soy broth- trypticase peptone 17 g, phytone peptone 3 g, NaCl 5 g, dipotassium phosphate 2.5 g and Bacto dextrose 2.5 g; nutrient broth- Bacto-beef extract 3 g and Bacto peptone 5 3. Preparation of inocula The soil inoculum was aseptically transferred into 10 m1 of sterilized T88 and incubated aerobically at 30° C. After about 3 days growth, 1 ml of this culture was transferred aseptically into another TSB tube and again incubated aerobically at 30° C until the tubes were inoculum as needed. Every two weeks these "stock cultures" were reinoculated into a fresh medium and grown at 30° C and then stored in the refrigerator until use. This process was repeated as needed through the experimental period. Experimental culture conditions Isolates were grown in culture tubes sealed with butyl rubber septa (Hungate tubes, Bellco Glass, Vineland, N.J.). The tubes con- tained 10 ml of 3% TSB plus either 3.5 mM KNO N O or 02 (air) as the 3’ 2 terminal electron acceptor. For anaerobic incubations the air was removed by evacuating and filling with He via needles connected to a manifold which was linked to a vacuum pump and He tank. The flushing cycle was repeated four times with a vacuum of -30 inches Hg achieved for 15 min each cycle. When N20 was required the desired concentration (generally 0.2 atmosphere) was added by syringe after first removing an equivalent volume of He by syringe. Acetylene was added where indicated at a concentration of 0.1 atmosphere. The tubes were then autoclaved at 121° C and 15 psi for 15 min. The tubes were inoculated with 1 m1 of the refrigerated inoculum. Tubes were incubated inverted to reduce chances of 02 leakage through the septum, and placed on a rotary shaker operating at 120 rpm. The incubation temperature was 30° C. The incubation period was one week for the nitrate dependent growth and N 0 concentration experiments and 2 until visible growth for the survey experiment. If no growth was visible the tubes were incubated for two weeks before analysis. Analyses Turbidity was measured as percent transmission at 660 nm in a Turner Spectrophotometer, Model 330. For growth yields a standard curve of cell dry weight versus optical density (optical density 8 2- loglo percent transmission) was used to determine biomass. The composition of gases in the sealed cultures was determined by gas chromatography. The culture was vigorously shaken by hand to ensure equilibration of the gas between the soluble and vapor phase prior to sampling. A sample, usually 0.5 ml, of headspace gas was removed by a 1 ml plastic syringe fitted with a Pressure Lock valve (Precision Sampling Corp., Baton Rouge, LA). The sample was injected into a Carle Model 8515 gas chromatograph ( Carle Instruments, Fullerton, CA), equipped with Poropak Q (3 mm x 1.8 m) and Molecular Sieve 5A (3 mm x 1.8 m) columns connected in series by a column switching valve. The detector was a microthermistor. The column temperature was 45° C. The carrier gas was He at a flow rate of 25 ml/min. Peaks were recorded on a chart recorder and were quantified by a computing integrator (Autolab I, Spectra-Physics, Santa Clara, CA). In the survey the integration value in u volt-sec is recorded to give an indication of the size of each peak since precise quantitation of each component was not necessary. Growth yield experiments The reference strains of A; faecalis, E; perfectomarinus, P; stutzeri, and Paracoccus denitrificans were used to determine growth yields on N20 and 02 as terminal electron acceptors. Inocula were grown aerobically on 3% TSB and then transferred (4 ml) to side-arm Lrlenmeyer flasks (164 ml) which contained 100 ml of 3% TSB. The flasks were sealed with rubber stoppers pierced by a glass tube capped with a serum stopper for sampling of headspace gas by syringe. The flasks were made anaerobic by evacuation and filling with He as des- cribed above. N20 was added to achieve a gas composition of 0.2 atmosphere in the manner described above. Flasks were incubated at 30° C on a rotary shaker at 150 rpm. Growth was measured as percent transmission in the side-arm tube at l or 2 hour intervals. This value was converted to cell dry weight by means of the standard curve for each organism. At the same time a sample of flask atmosphere was analyzed for N20 by gas chromatography. The total N20 content was determined for the vapor plus solution phases using a Bunsen coefficient of 0.67 Smith gt 21. (28). The same procedure was used for growth yield experiments with O2 and nutrient broth as culture components. Use of N20 as electron acceptor in MPN tubes The soils used are described in Table 1. After collection the soils were passed through a 5 mm sieve without drying and were stored in sealed plastic bags at 2° C until used. These soil samples consisted of six subsamples that were freshly collected from the upper horizon of the soil. The same soils were used in other MPN studies of denitrifiers but using different methods so that my results could be directly compared (N. V. Caskey, personal communication). The first dilution was prepared by blending 10 g of soil in a sterilized Waring blender for 2 min with 90 ml of sterilized distilled water containing 0.85% NaCl. One drop of Tween 80 was added per liter of the distilled water before sterilization. Ten-fold dilutions of the 5011 samples were prepared. One—tenth milliliter of the appropriate 10 Table 1. Characteristics of soils used. % Organic Series Texture Classification pH matter Brookston Loam Typic argiaquoll 7.6 3.2 Miami Sandy loam Typic hapludalf 6.6 2.7 Spinks Loamy sand Psammentic hapludalf 6.4 1.5 dilutions of the soil samples were transferred to each of the five Hungate tubes which contained 10 m1 of sterilized 3% TSB and 0.5 atm N20. The tubes were incubated on a rotary shaker at 30° C and observed daily for turbidity. After 14 days incubation, 0.5 ml of headspace gas was analyzed for disappearance of N20 by gas chromatography beginning with the tubes showing turbidity at the highest dilution. RESULTS AND DISCUSSION Incubation conditions In Gambles' (11) previous study and from other experiences in the laboratory it was noted that nutrient broth did not always support luxurious and consistent growth of denitrifiers. Therefore, in a preliminary study nutrient broth was compared with tryptic soy broth for support of growth of a variety of denitrifier strains. At the same time the ability of each strain to grow anaerobically on tryptic soy broth and N03- was also examined. The results for each of 123 strains on each medium is recorded in Appendix A. The results are summarized in Table 2. Tryptic soy broth was superior as 98% of the cells grew in this medium while only 75% grew in nutrient broth. Fifteen of the isolates lost their viability as they could not grow aerobically in any of the media while 10 of them could grow aerobically but had lost the ability to grow by denitrification. To further examine whether TSB was a better medium for growth of the denitrifiers, growth rates of P; perfectomarinus, P; stutzeri and A; faecalis on TSB were compared with those on nutrient broth under anaerobic conditions with 20% N20 as the electron acceptor. Both rate of growth and N20 use were much faster in TSB than in nutrient broth 11 12 Table 2. Comparison of complex media for supporting growth of denitrifier strains. Percentage of viable Number Number that a Medium that grew did not grow isolates that grew Nutrient Broth + 02 84 39 76 Tryptic soy broth + 02 108 15 98 Tryptic soy broth, 3.5 mM KNO3, no 02 98 25 89 a . Inoculum was pregrown on nutrient broth and tryptic soy broth; 110 of the 123 strains taken from the soil stock culture grew on one or both media. 13 (Table 3). The improvement in growth rate was 40 to 100% by use of TSB for the strains examined. Because of the nutritional and physiological differences among denitrifiers, it is not surprising that a single medium is inadequate for their cultivation or enumeration. Tryptic soy broth differs from nutrient broth in that it provides a readily utilizable carbon and energy source (glucose) a plant rather than an animal-derived protein, more total carbon and possibly more growth factors. Whatever the explanation, it would appear that the organic substances in nutrient broth were not adequate to satisfy the nutritional demands of sizeable portion of the denitrifying microflora. Marten (21) has also found that 0.3% Bacto-tryptic soy broth (Difco) solidified with 1.5% agar to be as good as a soil extract based medium for isolation and enumeration of total aerobic bacteria. Thus, a TSB based medium appears adequate for growth of soil denitrifiers in this collection and was the medium of choice for the denitrification study. Because all denitrifiers and aerobes prefer 02 over nitrogenous oxides as their electron acceptor, it was necessary to ensure that oxygen contamination could be minimized thus assuring the result was due to nitrogenous oxide dependent growth. Oxygen contamination can result from incomplete air removal during evacuation and flushing, possible air leakage through the septum, introduction of oxygen with needle and inoculum solution, and impurities in the gases, especially N20 which often contains 0.5 to 1% O . The adequacy of the procedure 2 used to minimize the influence of contaminating oxygen is demonstrated in Table 4. The Becker strains (Pseudomonas sp.) are denitrifiers which have lost the ability to reduce N20 to N2. They can grow with l4 3 .2 o; .5 2m 3383 4m :3 S A: N; 02 Rm wilumfifim 4m was 03 ed o.~ 2N «mm 3335803qu J mom mmh zooms mmH suoun mmH mcwmuum umnaac mo mm: xn mafia ucmfiuusz ucmauusz samuum coaumumcmw ca unm6m>ouaefi ALV A£\Hoenv unmoumm mafia :ofiumumcmu cowummaafius oNz .uouamoom couuomam HmafiEumu haco ozu mm omz Lufi3 Ammev nuoun >om ofiummuu mam nuoun unmwnuoa cfi mwamommm dfl mom “mononum 4M .mocHMmEOuowmumm 4M mo :uBOHw mo mumu mo comfiumaaou .m maan 15 nuSOHw on u o mxuwofinuSu kn :3onm nuBOHw u + m + o + N2 cu mmHMHuuwsmm fiumNuSum.dM + o + N2 ou mmfiwwuufiamw mcmommuooaw 4M. o o + maoumm mumwfiano Acflmuum .q magma ucmmcmmmm o 16 02, N03_ and N02-. The NTA strain is an obligate aerobe. Since no strains grew under He and the first three did not under He + 20% N 0, oxygen contamination was judged insignificant. Lack of growth also confirms that the medium does not contain other electron acceptors (eg. N03- or N02-) that could support growth. The medium was adequate for denitrifier growth as shown by growth of §;_fluorescens and 3;. stutzeri with N20. The first three organisms were routinely used as controls for oxygen contamination in other experiments. Initially 20% N20 was used to minimize any toxic effect that might be due to a highly water soluble, oxidizing gas. However, when N 0 gas 2 concentrations from 10 to 100% were used, cell yield increased in a linear manner (Figure 1). Thus toxicity is not apparent. Furthermore, final cell yield appears limited by the amount of N 0 available. Since 2 the yield is suboptimal at 20% N20, higher concentrations of N 0 are 2 recommended for future work. Distribution of N20 production and utilization capabilities among denitrifier strains Eighty-eight strains which grew by denitrification in TSB (Table 2) were examined for their ability to grow on N20 as their only electron 20 from NO3 1n the absence and presence of acetylene. The results for each strain are acceptor, as well as for their tendancy to produce N shown in Table 5 with summaries following in Tables 6-8. The viability of each strain under aerobic and denitrifying condition was also noted by observing turbidity in the presence of O2 and N03-, respectively, as terminal electron acceptors. Eight strains did not grow with O2 and three more did not grow with N03 . Thus 77 strains were confirmed as active denitrifiers for this survey (Table 6). Figure 1. 17 Growth of denitrifiers in different N20 concentratixan. JHCERIIEILI 025 020 .0 01 FINAé OPTICAL DENSITY ES 005 18 l l l d2 d4 d6 CONCENTRATION OF N20 (AIMS) Figure 1 08 l9 < + + +++ Om OH OOm H as mm mm H O + O O Om 1: HM O qu NO Hm n NOH :« uncommon peanuwmucH : o N .o .o memmme ua< -moz oNz ~z o~z mmmmmwwmmmmmmmw we“ «maumUM~Mua -M N camps” “mumsH” zuzoum oHOHmH> ocmeuoom mmHONHODQmoom Ocuamuuc 0 mm o 2 co Luzouo no mm 02 co Luzouu m mOumHomH uoHOHuuHcOO ox up O N 2 ma oz :0 zmzouo .mmuSuHsu oucouowou m can we coHuosvouO Ocm coHumuHHHuO .m «Hams 2() < + + ++ me OOH OOm O O OO OO : NOH OH oncoamou noumuwwucH m N N N N N N «.33 :< - oz 0 z z o z z o z 3235...“ o 2 .H N .02 .oz mazzumm OOJOOO oHOHmH> mcmHmuwom maHm noumuuum Hmcmeuwom ocv “cumuuum a :HmHum “mumaHO a ma 0: co nuao~u Ocuaaoom m moz :o nuzouo mm vwscHucou .n oHOwH mm ONz co numouo 21 O + + O OO O OO OOH OOH OO OOH OH < + + + OO O OOH NO ONH OO OOH OH < + + + u: u: OO uu OHH OO ONH I: < + + + O OO OOO O O HO NNH OH O O O O NO O HO uu NNH OO OOH OH O + + O OO OOH OO HOH ONH OO NoH O < + + +1+ OO O NOO O O OO OOH N O + + O OO H OHH OO OOH O < + + +++ HO NOH OHO O O OO OOH O < + + ++ u: u: u: u: nu OO OOH I: O + + O OO O OHO O OOH OO HOH O m + O O as u: NN O NOH OO OOH N < + + +++ OO OH OHO O O OO OOH N m + O O OO O NO O OOH OO HOH O m + O O OO uu OO O OOH OO OOH O O + O u: as OO O OOH OO ONH O O + + O a: u: ON O OOH NO HOHH O < + + + OO NOH OOO H O NO HHH 3 NOH OH wmcoamwu poumquucH O N N N N. N O N cum: uH< u 02 O z 2 O z 7 O z :HchEmu O 2 H N .02 .oz ammnumm zuaomm‘oHnHmH> wcwHOuwow msmm uowmouom HucuwquOm ocv poumwoum um awwuum OvumzHO wmm Ooz co cuzouu vwscHucou umuamoow no mm Oz co nuaouu .m magma N mm O 2 co OOJOOO 22 OosuoOumO Oman 0: .uc unauoouwv womuu .uu amazuma mo coquHuommv you OxOO 0mm < + +++ +++ OO OOH OOO nu OH ON < + + +++ H uu HOO O nu OO OOO < + + +++ OO OOH OOO Nu OO OOO IIIIIIII.II Humuusum .O < + + ++ u: u: u: O: O NO NOO IIIIIIIIIII mnuuoUMOOO < + + +++ O: u: OHH N O OO OOO .II smocstuww .O < + + +++ Dc u: NHO O O OO OOO IIIIIIIIIII ll mcmomwuoaHO .O O O O O OH O u: u: NOH OO OOO IIIIIIIII.II ocHooOcoE .O O + + O NO NO NO OOH OOH OO NOO mcwHomOouuam .O < + + ++ OO OOH NHO uu O OO HOO mamOHOHuOchO 4M < + + ++ OO O HON O O OO OON N O O O O NO O OO O OOH NO NON N < + + + NO N NOO O O OO NHON O < + + ++ u: us OON nu OO ON ONN O < + + + NO NOH HOH OO NO OO OON N < + + + OO NNH HOO N O OO OON 3 NOH CH Umcoamvu “OumuwwucHlllllllllllIIIII N uH< OOz ONz Nz ONz Nz ONz OchHnEmu O z N N .02 .oz Oum: I Namzuem OOJOOO uHOOmH> mcwHOuuom msHO usumuuom chmHOumum ocv uc Ouoom In chuOm umumsHO w mm 02 co Luzouo HOOOMUOM 0 mm 0 2 co canouo mm OOz co cmzouo Omchucoo .O mHOmH 23 Table 6. Number of denitrifier strains showing the indicated denitrification pattern. Description of denitrification Number Of a pathway used by isolates denitrifiers 1. Produce and utilize N20 (A) 64 2. No N 0 production and utilization but re uction of N03 to N2 (B) 1 3. Produce N20 but do not utilize it (C) 12 4. Reduce N03- + N2 but do not grow on N O (D) 5 2 5. Produce N20 in the presence of acetylene 66 6. Lost ability to denitrify and/or not viable (E) 11 7. Able to use N03- as a terminal e-acceptor [A+B+C] 77 8Total number of organisms studied was 88 24 Confirmation of ability of the denitrifiers to utilize N20 was based on the following:- 1. Partial or total disappearance of N20 when grown on N20. 2. Increase in N2 when grown on N03 . 3. Visual turbidity when grown on N20. 4. Accumulation of N20 from N03- in the presence but not in the absence of 0.1 atmosphere of acetylene. 20 from N03- in the absence of acetylene and a subsequent increase in N2. 5. Partial or total disappearance of N 6. Increase in C02 production when grown with N20. Results in Table 5 show some variability in extent of N20 reduction and growth. This was partially due to my collection of data after the appearance of turbidity, but not necessarily at the same stage of growth. Nonetheless, all of the above criteria could easily be dis- tinguished and gave a consistent interpretation for 69 of the 77 strains. For the remaining eight strains it was turbidity that was not clearly discernable. In these cases the tubes were scored as positive for N20 use if the concentration of N20 had diminished significantly. In these cases the limited growth also limited N20 reduction. Despite the first impression of variability of data, the number of clear-cut conclusions was high. Sixty-four or 83% of the 77 strains reduced N20 (Table 6), while 13 strains produced but could not use N20. One strain (No. 204) exhibited reduction of N03- to N2 but did not produce or use N20 suggesting that N20 may not be a freely diffusable intermediate in this 3 to N2 but did not have the capacity to grow on N20. This is apparently because case. Five strains (Nos. 42, 44, 69, 110, 151) reduced NO 25 these strains lack the capacity for phosphorylation associated with the N20 reduction. The fact that most strains which reduce N20 can also grow on N 0 suggests that the capacity for N20 reduction and phosphoryla- 2 tion are generally linked. One of the following hypothetical schemes can be assigned to each of the strains from the data in Table 5. N O -——-—-€>‘N2 fr m N O 2 A. N03" ——9 N02” -—————-> N0 ———> B. N0'————>No'—-—-——->No——————>N20———————>N2 3 2 £1 ATP? 20 C. N0 " ———-—;‘~NO 3 - -—-————€> NO -——-+——O> N I D. N03_ ———————> N02- ———-—> NO —————> N 20 -——*———> N2 ? 2 N-..) O o——————,>N 2 2 11? N20 ATP ? '2 ? E. N03'—--x—-—->N02‘—————>N0—————->N O—-—————————>N2 N20 Pathway A shows that N20 is a freely diffusable intermediate while B represents no production and utilization of extracellular N O by 2 denitrifiers. C represents production but no utilization of N20. Here the end product of denitrification is N20. D represents reduction of N03- and perhaps N20 to N2 but no growth occurs on N20 since ATP is not generated. E represents those cells which have lost their ability 26 to grow. All strains unable to grow on N03 were also unable to grow on N20. The numbers of denitrifiers fitting the above schemes are summarized in Table 6. Table 7 shows the number and percentage of strains in major taxonomic clusters capable of N20 utilization while Table 8 indicates the percentage of major species identified by conventional means that utilize N20. Unfortunately many of the groupings had too few strains to draw a conclusion on correlation of N20 reduction capacity with phenotype. For most groups 2/3 or more of the strains reduced N20. The most noteable exception is_§; aeruginosa. None of the strains clustered as this species grew on N20 (Table 7). The one strain classified as P; aeruginosa that did grow on N 0 (Table 8) was a 2 reference strain originally obtained from W. J. Payne (Gamble, 12). The absence of growth on N20 by P; aeruginosa is supportive of the same observation noted by W. P. Payne and J. L. Ingraham (personal communica- tions to J. M. Tiedje). A11 strains of P; stutzeri studied utilized N20 as a terminal e-acceptor. No strain of Pseudomonas sp. type 2 utilized N20. 3; fluorescens, biotype II was the most commonly encountered deni- trifier in the world-wide survey conducted by Gamble 35 El. (12). A high proportion of these strains reduced N 0 (Table 7) which suggests 2 that the capacity for N20 reduction exists in most soils. For several strains the presence of acetylene did not cause the dramatic increase in N20 expected. It is not clear if this is due to ineffective inhibition by acetylene of some strains or whether the sampling was premature. In the absence of acetylene N20 produced by reduction of N03- persisted in some of the tubes for only short periods. 27 Table 7. Distribution of the capacity to grow on N 0 among the similarity clusters of denitrifier strains found by Gamble 33 El. (12). Total number Total number Denitrifiers Cluster Probable identity of denitrifiers Of N20 that can number of cluster studied utilizers utilize N O (7.)a 2 l P;_f1uorescens 39 33 85 2 Pseudomonas sp. 6 6 100 3 _P; aeruginosa 2 0 0 4 P; aeruginosa 2 0 0 5 Pseudomonas sp. and Alcaligenes sp. 4 4 100 6 Pseudomonas ? l 0 0 7 Pseudomonas sp. 1 l 100 8 Pseudomonas sp. 2 2 100 9 Flavobacterium sp. 2 2 100 10 Ungrouped isolates 10 10 100 11 Reference cultures _§ _§. 75 Total 77 64 83 a . . Percentage based on Viable isolates. 28 Table 8. Major species that utilized N 0. 2 Percentage of N O a Number of Number utilizers in Species N20 utilizers studied number studied 1. P;_fluorescens II 20 25 80 2. A;_faecalis 3 4 75 3. Pseudomonas sp. type 2 0 5 0 4. Pseudomonas sp. type 4 l l 100 5. P;_aureofaciens 2 3 67 6. Pseudomonas sp. type 5 2 3 67 7°.B; aeruginosa 1 3 33 8. Flavobacterium sp. 3 2 67 9. §;_stutzeri 4 4 100 10. P;_fluorescens (?) '_4 ‘_4 100 Total 40 54 a Tentative identification given by Gamble (11). Percentage based on viable isolates. 29 This observation is in harmony with previous studies by Blackmer and Bremner (5) where they found that microorganisms accumulated N20 for a short time and subsequently reduced it to N2. N20 also accumulated temporarily and then was converted to N2 in both soil and microbial culture experiments (10, 24). This survey shows that 83% of these isolates produce and use N20. N20 was the end product of denitrification for 17% of the isolates. In concluding it is likely that soil can be a sink as well as a source of atmospheric N20 because of the high numbers of denitrifiers that can utilize and produce N20. The percentage of N20 users may have even been higher if these cultures were freshly isolated, since some of them may have lost the ability to synthesize N 0 reductase during their 2 period in the laboratory. This argument is supported by literature records which suggested that at least for fresh isolates from soil, essentially all reduce N O to N2 (24). Also Garcia (13) working with 2 soil showed a high correlation between denitrification rates measured by Warburg and N20 reduction. Gamble (12) also noted that a large percentage of his fresh isolates which originally denitrified no longer produced N2 gas after subculturing. Growthgyields Koike and Hattori (18) have reported that nitrate respiration is about 40% less efficient than aerobic respiration (4.5 vs. 7.5 g cells/mole glutamate with N03 vs. 02, respectively). Though I did not determine growth yield for 02 as the electron acceptor, the aerobic 'generation time was less than with N20, 0.5 vs. 1.0 hour on 0 vs. N O, 2 2 respectively, by A;_faecalis in TSB and 0.8 vs. 2.0 for P; perfectomarinus 30 in nutrient broth. This was a 50-60% reduction in growth rate due to N20. The above authors found a greater reduction in growth rate by denitrification, 1/5 to 1/7 of that with 02. Koike and Hattori (19) also reported that E; denitrificans showed identical cell yields per electron transferred when N03-, N02- and N20 were electron acceptors. Their data are summarized in the lower half of Table 9 to facilitate comparison with my data. Plots used to obtain my data are in Figure 2. Their data does not include maintenance energy which becomes more significant as growth rate decreases. The A; faecalis strain, which grew very quickly, had a yield similar to their values. The other strains had lower growth yields. The two with the lowest yield also had the slowest growth rate. Thus a large maintenance energy cost may be at least partially responsible for the lower yields. Use of N20 as an electron acceptor in MPN tubes There is no reported attempt to use N20 as the terminal electron acceptor in the enumeration of denitrifiers by the MPN procedure. This approach has the advantage that only denitrifiers can reduce N20 to N2 under these growth conditions. Thus this method would be specific for denitrifiers, a feature not found in currently used methods. Other methods measure disappearance of N03- and N02-. Problems are false positives due to dissimilatory nitrate reducers and the sometimes slow reduction of N02- possibly due to its toxic effect. A potential problem of the N20 reduction approach was the uncertainity as to how many denitrifiers could grow on N20. This concern has been alleviated by the finding that 4/5 of the denitrifiers surveyed could reduce N20. This error would be encompassed by the statistical error inherent in the 5-tube MPN method. Use of TSB with N20 would retain the nutritional .mmmoapsa m>Humummaoo How OOOOHUOH mH Ocm NNH .Hu0uumm Ocm mxHon muauHso soumn OH mcov omHm mmz mHmmnuamumO OH msHm> .OUCOCOOOHma pow Omuomuuoo mp uoc OHsou Ocm musuHso nouns OH ocoO mm: Onsum OE Eoum mumO “OOOOHUOH uo: Owumcm mocmcmuaHmz m 31 O; M; 823:8 oNz o . m a .3 32.328 INoz 3.2 NO OON 333:8 Imoz «OH 4.83% a 3:3. Eat! mcmonHuuHcmO .m ON N.N O3 92. oNz 9538303me J. N.N o.m H6 m2. oNz memoflflflcmv maoooomumm N.H H.O N.O OOH oNz Hummus; NM 0; 03 NS OB oNz 3383 ad HOV HImNOHHmo OO Huouamoom Hoe\mHHmo Ov wousom NOOOmoom :Hmuum mEHu Omuumwmamuu aouuomHm OHOHO OOBouO conumO couuomHm coHumumcmO awn OHwHw .uouamoom :ouuomHm mm ONz co OHDOHOU nouma OH GBOHO amSB mchuum umHOHuuchO Hmum>mm mo OOHOHO nusouo .O mHOmH 32 Figure 2. Growth yields of four denitrifier strains with N20 as terminal electron acceptor. MILLIGRAM DRY WEIGHT 0F CELLS 20 IS 33 J J 0.5 l I .5 ,, MOLES N20 CONSUMED Figure 2 34 advantages of TSB, decrease in vitro competition due to simultaneous growth of NO3 reducers, and simplify the requirement for a positive test for denitrifiers by simply determining partial or total disappearance of N20 from culture tubes. Therefore the use of the MPN procedure to enumerate denitrifiers capable of N20 utilization was tested with samples of three soils. The results were unexpected. No disappearance of N20 was observed in any tube, even at dilutions of 10'.3 and 10_4/g. Other methods had shown at least 106 organisms/g in these soils. The cause of the lack of N20 use could be due to absence of enough metabolizable carbon after faster growing aerobes and fermenters had used up the original substrate. It could also be due to the inability of cells to synthesize N20 reductase under these conditions. To discover the reason for this behavior I added fresh filter sterilized TSB + N03- (concentrated) to half of the tubes and TSB only to the other half. The additional carbon should have overcome any energy limitation and the N03- could serve as an inducer. However, no N20 disappearance was again observed after one week. In some tubes with added N03- a larger N2 noted indicating that organisms capable of reduction of N03- to N20 were present and active. In the survey (Table 6), pure cultures grown 0 peak was in the same medium commonly reduced N20 to N2. The difference between the MPN and pure culture results is puzzling. LITERATURE CITED 10. ll. 12. LITERATURE CITED Allen, M. B., and C. B. van Neil. 1952. Experiments on bacterial denitrification. J. Bact. 64;397-412. Bates, D. R., and P. B. Hayes. 1967. Atmospheric nitrous oxide Planet. Space Sci. l§;lB9-197. Beijerinck, M. W., and D. C. J. Minkman. 1910. Building and yerbrauch von Stickoxydul durch Bakteren Centr. Bakt. Parasitenk; 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: 359—364. Blackmer, A. M., and J. M. Bremner. 1976. Potential of soil as a sink for atmospheric N20. Geophys. Res. Lett. 3 No. 12, 739-742. Crutzen, P. J. 1972. A threat to the earths ozone shield. Ambio Crutzen, P. J. 1974. Estimates of possible variations in total ozone due to natural causes and human activities. Ambio 3:201- 210 O Crutzen, P. J. 1975. Physical and chemical processes which control production, destruction and distribution of ozone and some other chemically active minor constituents. (GARP) Publication series (WMO, Geneva, Switzerland), pp. 235-243. Delwiche, C. C. 1956. in Inorganic nitrogen metabolism (McElroy E. D. and Class B. eds), pp. 233-256, John HOpkins Baltimore. Delwiche, C. C., and D. E. Rolston. 1976. Measurement of small nitrous oxide concentrations by gas chromatography. Soil Sci. Soc. Am. J. 49:324-327. Gamble, T. N., M. R. Betlach and J. M. Tiedje. 1977. Numerically dominant denitrifying bacteria from world soils. Appl. Environ. Microbiol. §§}926-939. Gamble, T. N. 1976. Commonality of numerically dominant deni- trifier-strains isolated from various habitats. M.S. Thesis Michigan State University. 35 13. 14. 15. 16. l7. l8. 19. 20. 21. 22. 23. 24. 25. 26. 36 Garcia, J. L. 1974. Reduction de L'oxyde nitreux dans les sols de rizieres du senegal: measure de L'activite denitrifiante. Soil Biol. Biochem. 6:79-84. Gayon, V., and G. Dupetit. 1882. Sur la fermentation des nitrates. Compt. Rend. Ser. 25:644-646. Gayon, V., and G. Dupetit. 1882. Sur la transformation des nitrates en nitrites. Compt. Rend. Ser. 95:1365-1367. Gayon, V., and G. Dupetit. 1886. Recherches sur la' reduction des nitrates par les infinimentpetits. Soc. Sci. Phys. Nat. Bordeaux Ser. 3:201-307. Hauck, R. D., and S. W. Melsted. 1956. Some aspects of the problems of evaluating denitrification in soils. Soil Sci. Soc. Am. Proc. 20:361-364. Koike, 1., and A. Hattori. 1974. Growth yield of a denitrifying bacterium Pseudomonas denitrificans under aerobic and denitrifying conditions. J. Gen. Microbiol. 88:1—10. Koike, I., and A. Hattori. 1974. Energy yield of denitrifica— tion: An estimate from growth yield of continuous cultures of Pseudomonas denitrificans under nitrate-nitrite, and nitrous oxide limited conditions. J. Gen. Microbiol. §§;ll-l6. Kluyver,_A. J., and W. Verhoeven. 1954. Studies on true dissimila- tory NO reduction II. The mechanism of denitrification. Antonie van Leeuwenhoek. J. Microbiol. Serol. 20:241-262. Marten, J. K. 1975. Comparison of agar media for counts of viable soil bacteria. Soil Biol. Biochem. 13401-402. Matsubara, T. 1971. Some properties of the N20 anaerobically grown cell. J. Biochem. 69:991-1001. McElroy, M. B., J. W. Elkins, S. C. WOfs, and Y. L. Yung. 1976. Sources and sinks for atmospheric N 0 Rev Geophys. Space Phys. 14: 143-150. - 2 '— Payne, W. J. 1973. Reduction of N20 by microorganisms. Bacteriol. Pinchinoty, F. and L D' Ornano. '1961. Recherches sur La reduction du protoxyde d'azote par Micrococcus denitrificans. Ann. Inst. Pasteur. 101;418-426. Sacks, L. E., and H. A. Barker. 1952. Substrate oxidation and N20 utilization in denitrification. J. Bact. 64:247-252. 27. 28. 29. 30. 37 Schutz, K,, C. Junge, R. Beck, and B. Albrecht. 1970. Studies of atmospheric nitrous oxide. J. GeOphys. Res. Z§;2230-2242. Smith, M. S., J. 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. 425611-615. Suzuke, S. 1912. Uber die Entstehung der. Strickoxyde in Deni- trification-Centr. Bakt. Parasitenk, Abt. 11,31:27-49. Wang, W. C., Y. L. Yung, T. Mo. Lans and J. E. Hansen. 1976. Green house effects due to manmade pertubations of trace gases. Science 194:685-689. CHAPTER II EFFECT OF SOIL pH ON DENITRIFICATION 38 INTRODUCTION Many bacteria are quite tolerant of acidity and are able to grow and develop over a wide range of H+ activity while others are restricted to either acid or alkaline conditions. Although it is generally assumed that denitrification is favored in neutral to alkaline habitats, few studies have been performed with the active species and with soils. Though there exists a considerable literature on denitrification, many reports on the physiological and ecological characteristics of deni- trifying microbes are contradictory. There are differences of opinion among investigators concerning the chemical and/or biological processes which lead to the production of nitrogen oxides. The size and activity of the denitrifying flora in different ecological circumstances are key factors in determining the rate of loss of nitrogen from soils but there is little known of the environmental factors regulating the abundance or activity of these microorganisms. The effect of acidity on denitrification may be exerted in a number of distinctly different ways and the sparse denitrifying popula- tion in an acid environment may be a reflection of an influence upon growth rather than an effect upon the denitrifying mechanism itself. Two ways of establishing the significance of pH to microbial deni- trification are by a determination of the effect of the H+ activity on the size of the denitrifying population in natural circumstances and by a characterization of specific organisms with regard to their capacity 39 40 to liberate dinitrogen at various pH levels. Studies with pure cultures will help to support the ecological investigations of the influence of acidity. However, since the conditions designed in the laboratory have to be quite different from those existing in the microenvironments within the soil, the application of these results is difficult. The comparison of the activity of denitrifying microorganisms in a liquid growth medium and after inoculation into sterilized soil is one approach to determine whether various H+ concentrations significantly affect the denitrifying potential of certain microbes. Recent work has provided indirect evidence that significant gaseous loss of fertilizer nitrogen can occur through chemodenitrifica- tion, i.e. by chemical decomposition of nitrite formed by nitrification of ammonium yielding fertilizers in soils (1, 2, 5, 10, 23). Most workers have assumed that the rate and extent of nitrite decomposition in soils increase with a decrease in soil pH because solution studies have shown that decomposition of nitrite is promoted by acidity. Studies on denitrification products show that acid conditions are more favorable for formation of N 0 and N0 than neutral and alkaline condi— 2 tions which favor N formation (9, 24, 26). 2 Nevertheless, there are differences of opinion among investigators concerning the nature and importance of chemical and/or biological processes which lead to the production of the nitrogen oxides. The aim of this project was to determine the effect of H+ activity on biological denitrification and the extent to which N O is produced 2 by enzymatic or chemical reactions. The objectives were therefore, (1) to determine the rate and products of denitrification by soil samples differing in pH; and (2) to determine the effect of pH adjustment on denitrification rate and products of the soil samples. SUMMARY OF PREVIOUS INVESTIGATIONS Earlier investigations have shown that denitrification is favored by a relatively low hydrogen ion concentration. Broadbent (6) reported that denitrification is favored below pH 7 where as other investigators (l4, 4) concluded that nitrogen loss was considerably suppressed under acidic conditions. In another report (16), it is concluded that no correlation between pH and denitrification parameters could be found. It is generally assumed that denitrification is favored in a neutral to alkaline ecological system and that denitrifying populations in otherwise optimal environmental conditions fail to release gaseous nitrogen at high H+ activities (18). Dawson and Murphy (12) have shown that denitrification rates give parabolic curves as a function of pH with a peak at 7.0. The rates at pH 6.0 and pH 8.0 were approximately halved. However, Wiljer and Delwiche (24) and Bremner and Shaw (4) have shown that the rate of denitrification increases linearly from pH 4, levels off between pH 7 and 8, then declines, though not ceasing until at pH 9.5. Neutral to slightly alkaline pH ranges not only effect faster rates of denitrifica— tion but also the complete reduction to N2. Bollag, gt 31. (3) concluded that formation of nitric oxide in acid soils was largely chemical since sterilized soils evolved as much nitric oxide as controls upon addition of nitrite. Reuss and Smith (19) found that small amounts of N2 and N20 are formed by decomposition 41 42 of nitrite in acid soils. They also indicated that the amount of nitrite formed increased with a decrease in soil pH and that soil sterilization has little effect on the amount of N2 or N20 formed by treatment of acidic soils with nitrite. Denitrification and chemical nitrite decomposition seem to be the two predominant processes in volatilization of nitrogen, but it is not clear which one of the two mechanisms is of greater practical importance. Some investigators hold the biological reaction of denitrification most responsible for nitrogen losses from the soil (18); whereas, other studies tend to emphasize more the chemical volatilization (17,7). There is little doubt that both processes are influenced by factors such as pH, organic matter and others. Bremner and Shaw (4) demonstrated that the type of organic matter, pH, temperature and the aeration are among the chief variables governing the rate and magnitude of nitrogen loss. Valera, gt 31. (22), found that regardless of seasonal changes the number of denitrifiers was found to be positively correlated with pH, the coefficient of correlation (r) ranging from 0.66 to 0.97. They also found that the size of both the denitrifier population and the total bacterial population was positively correlated with soil pH but that the denitrifying bacteria were more sensitive to acid environments than the bacterial microflora as a whole. On the other hand, in an investigation of Australian soils, Jensen (15) found no relationship between H+ activity and microbial number although he did note a positive correlation with organic matter content. In this study I have investigated phase II denitrification rates in Nigerian and Michigan soils which vary from strongly acid to neutral. The purpose was to determine whether biological denitrification occurs under acid conditions and whether denitrifying populations of difference acid tolerance might have developed in the various habitats. Phase II denitrification rates have been defined by Smith and Tiedje (21) as reflecting the amount of denitrifying enzymes that can be produced by the population of denitrifying organisms present in the natural soil. Thus the rates reported here are not rates expected in nature but reflect the potential of the indigenous population. MATERIALS AND METHODS .9211s. The collection of the samples involved taking six subsamples of fresh surface soil (0-15 cm deep, including litter layer) from an approximately 10 m2 homogenous area. The six subsamples were made into one composite sample. Approximately 0.5 kg of the composite sample was enclosed in a plastic bag sealed without drying and stored at 2° C. Nigerian samples were immediately shipped by air to the laboratory; all carried a non-sterilization entry permit. Five Nigerian soils ranging from acid to neutral pH were obtained for the study. Their major characteristics are summarized in Table 10. Samples 1, 4 and 5 were collected from a forestry reserve that had not been cultivated for over 50 years while sites 2 and 3 have experienced slight cultivation. No evidence of addition of any form of nitrogen fertilizer was indicated at any site. Samples were supplied with information on the crop grown, soil type, previous crops, approximate location, i.e., distance and direction from nearest geographical location, whether site was cultivated or not, drainage, mean rainfall and other useful information. 43 44 Table 10. Characteristics of Nigerian soils used in study. Nigerian soil pH Drainage NO3-N N02—N ------ ppm N ------ N1 3.8 Somewhat well drained 50.0 0.05 N2 4.4 Well drained 44.0 0.11, N3 6.3 Well drained 51.0 0.11 N4 6.7 Well drained 7.0 10.06 NS 4.5 Poorly drained 21.5 0.08 a Soil pH measured with a glass electrode pH meter (Beckman Model 4500 Digital pH meter using a 1:1 soil water suspension). 45 Acid soil samples were also obtained from experimental plots on the Michigan State University farm (courtesy of Dr. A. R. Wolcott, Dept. Crop and Soil Sciences). These plots have been receiving different carriers of nitrogen fertilizer since 1959 (8). One heavy textured soil was obtained with the help of Dr. Christenson from the Saginaw experimental farm. The characteristics of the Michigan soils are summarized in Table 11. The soils are coded N, Nigerian; W, Wolcott and SAG, Saginaw. Assay of denitrification Moist soil taken from the stored samples was passed through a 5 mm sieve and 50 g was placed in 125 ml Erlenmeyer flasks. Thirty milliliters of water were added to make a slurry and the flask was sealed with a rubber stopper. The acetylene inhibition method was used to measure the rate of denitrification Smith st 31., (21). No additional N03- was added. The soil was made anaerobic by evacuating and filling the flask three times with He. Acetylene was added by syringe to achieve a concentration of 0.1 atm after withdrawing an equal volume of He. The flasks were then incubated at 30° C on a rotary shaker operating at 250 rpm. All treatments were replicated three times. The headspace gas was sampled periodically by syringe to determine N20 (and C02) concentrations by a microthermistor detector after separation by gas chromatography as previously described (Chapter I). Quantitation of N20 was by a standard curve and included corrections for N20 solubility. Autoclaving and propylene oxide were investigated as methods to achieve sterile controls. The autoclave treatment was 30 min at 121° C, 15 psi, three times with intervals of at least 8 h between. The 46 Table 11. Characteristics of Michigan soils used in study. — a - 3 Plot pH Treatment NO3 —N NO2 -N ------- ppm-N ------- W1 5.8 Ca(N03)2 17.5 0.03 W2 5.0 (NH4)ZSO4 11.0 0.04 W4 5.9 Control 21.5 0.03 W8 6.1 NaNO3 14.0 0.03 w18-1b 7.0 Ca(N03) 18.5 0.05 WlS-l 6.7 (NH4)2804 4.5 0.04 W20—l 6.8 Control - - W13-l 7.0 NaNO3 19.0 0.03 SAG 7.2 - 9.0 1.41 a -N and NO -N determined by standard Technician Aqu Analyzer II procedure. Analogous to above treatments but recently limed. propylene oxide treatment was 2 m1 of propylene oxide dispersed over the 25 g of soil, sealed and let set for 2 days after which the flasks were opened in a hood to let remaining prOpylene oxide diffuse away. The effect of sterilization treatments on pH was determined by measuring soil pH in a 1:1 water slurry before and after treatment. RESULTS AND DISCUSSION Evaluation of sterilization methods The effect of the sterilization treatment on pH is shown in Table 12. The pH change due to autoclaving soil was insignificant (0.04 pH units) but pH was significantly changed by prOpylene oxide (increase of 0.8 pH units). This had also been noted by Skipper gt 31. (20). Both methods seemed to effectively sterilize the soil as measured by lack of CO2 production. Neither treatment stimulated N20 production. Because of the potential significance of acid catalyzed chemodeni- trification to this study, the propylene oxide method was rejected since the original pH could not be maintained in a sterilized control. Thus autoclaved soil was used as the control for chemodenitrification in the following studies. Denitrification rates in soils of different pH. The soil pH did not change substantially (<0.01 pH unit) during the short anaerobic incubation period (Tables 13 and 14). Apparently the soils had adequate buffering capacities to maintain their original 3 . was upward, probably reflecting the loss of the anion. pH despite the consumption of NO In all cases the slight pH change The denitrification rates of the Nigerian soils are summarized in Table 4. The soil of highest and lowest pH showed no denitrification. 47 48 Table 12. Effect of autoclaving and addition of propylene oxide on pH of Saginaw soil. pH AC02 ANZO Treatment Initial Final ApH (103 x IV.)8 1. Non sterile soil 7.62 7.66 0.04 3.7 2.9 2. Propylene oxideb 7.62 8.42 0.8 0.3 0 3. Autoclaved soil 7.62 7.61 0.01 0.4 0 a . Integration value; these treatments were incubated for 10 hours and in the presence of 0.1 atm of acetylene. b 2 ml added/25 g soil in flask. 49 Table 13. Rate of denitrification in Nigerian soils of various pHs. . . . a Rate of denitrification Soil Ingfiial Figal (nmol NZO-g soil-l'h—l) N1 3.8 3.88 0b N2 4.4 4.45 8 N5 4.5 4.55 6 N3 6.3 6.34 5 N4 6.67 6.71 0 a Incubation was for 13 hours in the presence of 0.1 atm acetylene and no 02. A slight increase in N20 was noted for the first hour only. 50 Table 14. Rate of denitrification in Michigan soils; soils are paired to compare unlimed and lime treatment. Rate of denitrification Soils Ingfiial Figal (nmol NZO-g soil-1°h-l) r2 W2 5.0 2 0.99 WIS—lb 6.73 11 1.00 W1 5.8 ml W18-1 7.01 7.05 15 W4 5.95 4 0.99 W20-l 6.8 7 0.85 WS 6.1 5. 6 m1 Wl3-1 7.00 7.04 15 SAG 7.2 7.26 55 a Incubation was for 13 hours in the presence of 0.1 atm acetylene and no 02. ‘ U Limed soil; the preceding soil is identical except unlimed. 51 Most interesting, however, was the substantial denitrification rate of the two acid soils, i.e. pH 4.4 and 4.5. Autoclaved soils showed no N20 production. Biological reduction of N03- to N02- could be followed by chemical decomposition of HONO. However, the product is primarily N0 and not N20, which was measured in this study. Thus, these results are interpreted to mean that acid-tolerant denitrification did occur in these low pH Nigerian soils. Similar studies were conducted with Michigan soils which had been decreasing in pH since 1959 due to regular additions of different N fertilizer salts (termed "carriers"). In this case the pH's were not as low (5.0 to 6.1) as the Nigerian soils. However, the denitrification rate was very low in all soils. The same soils which had been limed showed much higher denitrification rates. Thus, for the Michigan soils, it appears that acid tolerant denitrifier populations did not develop in the comparatively short period of acid conditions. The Saginaw soil, which is a much heavier textured soil, showed much higher denitrifica- tion rates than the other soils. This is expected since a denitrifier population as well as derepression of denitrifying enzymes would be expected in this more 0 limited habitat. 2 Considering the results of pH influence on denitrification one does get the impression that there are denitrifier populations which vary in their sensitivity to acidity. The data are consistent with adaption or selection of acid tolerant communities in soils which have been acid for long periods of time. Future work This study is only preliminary; substantial additional work is needed before a comprehensive picture of pH influence on denitrification 52 can be established. Ideas for future experimental work are itemized below: 1. 2. Isolation of acid tolerant denitrifiers. Determination of the ratio of acid tolerant to total denitrifiers in the soil. Alteration of the pH by addition of a base or an acid and determine the rate of denitrification of the soils under adjusted pH's. Use of selected pure cultures to support the ecological investigations on the influence of acidity. Use of autoclaving to sterilize soil and then carrying out more studies on possible chemodenitrification at low pH. LITERATURE CITED 10. 11. LITERATURE CITED Allison, F. E. 1965. Evaluation of incoming and outgoing process that affect soil nitrogen. In 8011 Nitrogen (W. V. Bartholomew and F. E. Clark, Eds.) pp. 573-606, American Society of Agronomy, Madison, Wisconsin. Allison, F. E. 1966. The fate of nitrogen applied to soils. Adv. Agron. 18:219-258. Bollag, J. M., S. Drzymala and L. T. Kardos. 1973. Biological versus chemical nitrite decomposition in soil. Soil Sci. 116:44- 50. Bremner, J. M., and K. Shaw. 1958. Denitrification in soil II. Factors affecting denitrification. J. Agric. Sci. 51:40-52. Broadbent, F. B., and F. E. Clark. 1965. Denitrification in soil nitrogen (W. V. Bartholomew and F. E. Clark, Eds.) pp. 344-359, American Society of Agronomy, Madison, Wisconsin. Broadbent, F. E. 1951. Denitrification in some California soils. Soil Sci. 125129-137. Bulla, L. A., C. M. Gilmour and W. B. Bollen. 1970. Nonbiolog- ical reduction of nitrite in soil. Nature. 225:66-70. Burutolu, E. F. A. 1977. Effects of long-term application of high rates of nitrogen carriers of soil acidity, exchangeable cations and soil organic matter. M.S. Thesis, Michigan State University. Cady, F. B., and W. V. Bartholomew. 1960. Sequential products of anaerobic denitrification in Norfolk soil material. Proc. Soil Sci. Soc. Am. 24:477-482. Cleemput, O. V., W. H. Patrick and R. C. McIlhenny. 1975. Nitrite decomposition in flooded soil under different pH and redox poten— tial conditions. Soil Biol. Biochem. 1:329-332. Cleemput, O. V., and W. H. Patrick, Jr. 1974. Nitrate and nitrite reduction in flooded soil at controlled redox potential and pH. Transactions Tenth International Congress Soil Science, Moscow..2:152-159. 53 12. l3. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 54 Dawson, R. W., and K. L. Murphy. 1973. In "Advances in Water Pollution Research (S. H. Jenkins ed.) pp. 671-680, Pergamon Press, Oxford. Focht, D. D., and J. Joseph. 1973. An improved method for the enumeration of denitrifying bacteria. Soil Sci. Soc. Amer. Proc. 31:698-699. Jansson, S. L., and F. Clark. 1952. Losses of nitrogen during decomposition of plant material in the presence of inorganic nitrogen. Soil Sci. Soc. Amer. Proc. 11:330-334.‘ Jensen, H. L. 1934. Contributions to the microbiology of Austra- lian Soils 1. Numbers of microorganisms in soils and their relation to certain external factors. Proc. Linnean Soc. News South Wales, 525101-117. Khan, M. F. A., and A. W. Moore. 1968. Denitrifying capacity of some Alberta soils. Can. J. Soil Sci. 48:89-91. Nelson, 0. W., and J. M. Bremner. 1979. Gaseous products of nitrite decomposition in soils. Soil Biol. Biochem. 2:203-215. Nommik, H. 1956. Investigations on denitrifications in soil. Acta Agr. Scand. 6:195-228. Reuss, J. D., and R. L. Smith. 1965. Chemical reactions of nitrites in acid soils. Proc. Soil Sci. Soc. Amer. 22:267-270. Skipper, H. D., and D. T. Westermann. 1972. Comparative effects of propylene oxide, sodium azide and autoclaving on selected soil properties. Soil Biol. Biochem. 55409-414. Smith, M. S., J. 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. Amer. J. _42: 611-615. Woldendorp, J. A. 1968. Losses of soil nitrogen. Stikstof No. 12: 32-46. Wijler, J., and C. C. Delwiche. 1954. Investigations on the denitrifying process in soil. Plant and Soil. 5:155-169. APPENDIX APPENDIX A. Capability of denitrifier isolates to grow aerobically on three different media. a a Nutrient broth, TSB, N03 TSB, Isolate number Isolate name 02 no 02 O2 4 §h_faecalis +b 0b 0b 6 P. type 2 + 0 + 12 P. type 2 0 + + 13 PL_f1uorescens I + + + 15 P;_fluorescens (7) + + + 17 .5; faecalis 0 0 0 18 A:_faecalis 0 +- + 20 £2_faecalis 0 0 + 21 §2_faecalis 0 + + 28 .5; faecalis 0 O + 30 'A;_faecalis 0 O + 31 §h_faecalis 0 + + 36 Unknown type 3 + + + 39 Unknown type 3 + + + 40 §&_faecalis + + + 41 Ah_faecalis 0 0 0 42 PL_fluorescens II + + + 43 ‘A; faecalis + + + 44 ._i fluorescens II + + + 45 §;_fluorescens II + + + 46 Flavobacterium sp. + + + 47 P;_fluorescens II + + + 49 P. fluorescens II + + + 51 P. type 2 + 0 + 52 .2; fluorescens II + + + 53 P;_fluorescens II + + + 54 P. type 2 + + + 55 P. type 4 + + + 56 P. type 2 0 0 + 58 P;_fluorescens II + + + 59 P;_aureofaciens II + + + 61 P; fluorescens II + + + 62 P;_aureofaciens II + + + 63 P; fluorescens II + + + 64 §;_fluorescens II + + + 65 A;_faecalis + + + 66 P;_fluorescens II + + + 67 P; fluorescens II + + + 68 P;_f1uorescens II + + + 69 P;_fluorescens II + + + 70 P;_fluorescens II + + + 71 P. type 5 + 0 0 72 fig; fluorescens II + + + 73 P;_fluorescens II + + + 55 a Isolate number 56 Isolate namea Nutrient broth, 02 TSB, NO no 0 2 3 TSB, 74 75 78 79 80 81 82 83 84 85 87 89 90 97 98 99 101 102 103 104 105 106 107 108 110 111 115 1181 126 129 133 141 143 144 148 149 151 153 154 155 156 163 164 167 171 172 174 175 type 5 fluorescens fluorescens fluorescens fluorescens type type type type type type fluorescens faecalis type 9 fluorescens faecalis faecalis faecalis type 10 faecalis fluorescens faecalis type 11(not Unknown type 3 P. type 12 GNO‘NU‘N apppspwppapwaaasaswppwa §;_f1uorescens (7) Unknown type 15 type 16 type 18 type 18 type 18 type 16 type 11 faecalis faecalis type 11 type 19 type 19 eutrophus type 20 aeruginosa type 11 aeruginosa aeruginosa faecalis type 16 P. type 16 >W'U’d’d'd'd FFFFPL wwwpal ? "U Flavobacterium sp. II II II II II II 11 fl ooo++o++++o++o++o++++o++++++Ooo+o+oo+o++o+o+++++ oo++++++++++++++oo+++O++++o+ooo+o+oo++++o+o+++++ oo+++++++++++++++++++O++++++O+++O+oo++++o+o+++++ 57 a a Nutrient broth, TSB, N03 TSB, Isolate number Isolate name 02 no 02 02 177 Flavobacterium sp. 0 + + 179 P. type 19 0 + + 183 ‘2; fluorescens IV + + + 185 §;_fluorescens IV + + + 188 P. type 11 + + + 189 Unknown type 21 + + + 190 §;_f1uorescens (7) + + + 191 A; faecalis + + + 192 Bacillus sp. 0 + + 193 Bacillus sp. 0 + + 195 ‘P; stutzeri + + + 196 §;_fluorescens (?) + + + 199 Unknown type 22 0 0 0 202 P. type 23 0 + + 204 Unknown type 24 0 + + 205 .2; fluorescens II + + + 206 .2; fluorescens IV + + + 221 §;_stutzeri + + + 224 §;_stutzeri + + + 2312 §;_stutzeri 0 + + 232 P. type 25 + o + 234 P. type 11 0 + + 991 P;_denitrificans + + + ATCC 13867 992 §;_aureofaciens + + + ATCC 13985 993 §;_mendocino 0 0 0 ATCC 25411 994 (A;_faecalis + + + ATCC 8750 995 P;_f1uorescens + + + ATCC 17822 996 §;_aeruginosa + + + 997 Pa. denitrificans + + + ATCC 2008 998 §;_stutzeri + + + ATCC 17588 + + + 999 P;_perfectomarinus + + + Pseudomonas sp. (Becker strain 388) +' + + Pseudomonas sp. (Becker strain 402) + + + Pseudomonas sp. (NTA strain) + 0 + a Isolate numbers are tentative identifications and were given by Gamble (12). b Growth indicated by turbidity (+) or lack of it (0) after incubation at 30° C for 14 days. MICHIGAN STnTE UNIV. LIBRARIES IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHII 31293100658016