THE COMMONAUTY 0F NUMERICALLY DOMINANT DENITRIFIER STRAINS ISOLATED FROM VARIOUS HABITATS Thesis for the Degree of M. & THOMAS NELSON GAMBLE 1975 3.? . it: ' l Eiiymz. 11...: . LIBRA RY Mulligan .3133 Umvers'ty '7 K 7 /Cali% 6 ABSTRACT THE COMMONALITY OF NUMERICALLY DOMINANT DENITRIFIER STRAINS ISOLATED FROM VARIOUS HABITATS By Thomas Nelson Gamble Soils, fresh-water lake sediments, and a nitrified poultry manure, were examined for predominant denitrifier species. The samples were from eight countries and inclu- ded rice, crop, rainforest, desert, acid, organic, and waste- treated environments. Denitrifier populations were gener- ally 105 to 106 organisms per gram dry weight. The ratio of population densities of denitrifiers to organisms which reduce nitrate only to nitrite to total organisms which can grow anaerobically was fairly constant among samples; the average ratio was 0.26 : 0.68 : l. A total of 1500 isolates which grew on nitrate agar incubated in an anaerobic glove box were tested for the ability to denitrify. Following purification, 147 isolates were confirmed as denitrifiers by the production of N O 2 and/or N2 during growth in nitrate broth. The remaining isolates either produced nitrite, ammonia, or could not be maintained in culture. The denitrifier isolates were characterized using 52 properties appropriate for the Pseudomonas - Alcaligenes group. Pseudomonas was the Thomas Nelson Gamble dominant genus, whereas Alcaligenes faecalis was the most commonly isolated species. Other denitrifiers isolated included: Pseudomonas fluorescens biotype II, Pseudomonas fluorescens biotype IV, Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas aureofaciens, and Pseudomonas solan- acearum. Strains of a denitrifying Flavobacterium species were isolated which is the first report of denitrification by a member of this genus. A few denitrifying bacteria in the genera Corynebacterium and Bacillus were also isolated. About one-third of the isolates do not appear to be closely related to any recognized species though many do conform to the genus Pseudomonas. These were grouped according to com- mon characters into 25 identifiable types. No isolates similar to Pseudomonas denitrificans were recovered. A high correlation between temperature of isolate growth and temperature of habitat was noted. All isolates from trop- ical areas (mean annual temperature < 20 C) failed to grow at 4 C while 67% grew at 41 C. In comparison 68% of the isolates from temperate soils grew at 4 C and only 9% grew at 41 C. In conclusion, members of the Pseudomonas and closely related Alcaligenes genera were found to km; numerically dominant since they represent 89% of the total denitrifying isolates. The species of greatest importance appear to be Pseudomonas fluorescens biotype II and Alcaligenes faecalis which were found in 58% of the samples and comprised 41% of the isolates. THE COMMONALITY OF NUMERICALLY DOMINANT DENITRIFIER STRAINS ISOLATED FROM VARIOUS HABITATS BY Thomas Nelson Gamble A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1976 to my grandmothers ii ACKNOWLEDGMENTS I am especially grateful to Dr. James M. Tiedje for his advice and patience throughout the course of this study. I also wish to extend my appreciation to the members of my guidance committee, Dr. Michael J. Klug and Dr. John Breznak. 'Special thanks are due to all of the sample contri- butors. I also appreciate the assistance of Noorjeham Bhimji, Judy Benedict, Marshal Brulez, and William Caskey. These investigations were supported in part by grants from the EPA - International Joint Commission, the Rocke- feller Foundation, USDA Regional Research, and the Michigan Agriculture Experiment Station. iii TABLE OF CONTENTS Page LIST OF TABLES C O O O O O O O O O O C C O O O O O 0 Vi LIST OF FIGURES O O O O O O O O O O O O O O O O O O Vii INT RODUCT ION O O O O O O O O O O O O O O O O O O O . O l MTERIALS AND WTHODS O O O O O O O O O O O O O O O 4 Description Of Samples . . . . . . . . . . . . 4 Sample Collection, Handling, And Storage . . . 5 Sample Characterization . . . . . . ... . . . 6 Isolation And Enumeration . . . . . . . . . . 7 Methods . . . . . . . . . . . . . . . . . . . 10 'Denitrifier Characterizations . . . . . . . . 11 Effect Of Phosphate In Media . . . . . . . . 16 Effect Of Tween 80 . . . . . . . . . . . . . . 17 Comparison Of Nitrate Broth With Soil Extract- Yeast Extract Broth . . . . . . . . . . . . . 18 RESULTS . . . . . . . . . . . . . . . . . . . . . . 19 Sample Information And Physical Measurements . 19 Medium Selection . . . . . . . . . . . . . . . 19 Effect Of Tween 80 As A Soil Dispersing Agent 24 Effect Of Phosphate . . . . . . . . . . . . . 24 Quantitation Of Total Anaerobes, Denitrifiers, And Nitrite Accumulators . . . . . . . . . . . 27 The Ability Of Isolates To Utilize Nitrate . . 31 Identification Of Isolated Denitrifying Bac- teria O C I O O O O O O I O O O O O O O O O O 3 3 Temperature Relationship . . . . . . . . . . . 37 iv DISCUSSION . Development Of A Medium And Method For the Isolation And Enumeration Of Denitrifying Bacteria Commonality Of Denitrifier Isolates Correlation Between Samples, And MPN's And Isolates Improvements LITERATURE CITED APPENDIX . O Page 43 43 46 49 50 51 55 Table 10 11 12 l3 14 LIST OF TABLES Characters Examined for Each Denitrifier ISOlate O O O I O O O O O O O O O O O O O O O 0 Summary of Information on the Samples Provided by Contributors or Determined Experimentally .. The Effect of Phosphate Buffer on the Growth and Gas Production of Selected Soil and Sedi- ment organisms O O O O O O O O O O O O O O O 0 Population Densities and Ratios of Denitrifiers, Nitrite Accumulators, and Total Anaerobes . . . Percent of Isolates Utilizing Nitrate and Producing the Following Nitrogen Compounds . . Denitrifiers Isolated From Samples . . . . . . Major Genera Recovered . . . . . . . . . . . . Major Species Recovered . . . . . . . . . . . . Samples from Tropical and Temperate Locations . Percentages of Sand, Silt, and Clay in the Soil Samples as Determined by the Hygrometer Method. Results of 52 Properties Tested . . . . . . . . Names of the Denitrifiers . . . . . . . . . . . Number and insertion of Flagella of Selected Denitrifiers . . . . . . . . . . . . . . . . . Samples, Contributors, and Their Addresses . . vi Page 12 20 25 28 32 38 40 41 41 55 58 67 69 7O LIST OF FIGURES Page Scheme Utilized for MPN, Isolation, and Confirmation of Denitrifying Bacteria . . . . . 8 Characterization Scheme for the Identification of Soil and Sediment Denitrifiers . . . . . . . 35 vii INTRODUCTION Denitrification is one of the most important processes in the nitrogen cycle since it is the means by which com- bined nitrogen is lost to the biosphere. It is defined as the biological reduction of inorganic forms of nitrogen (ni- trate and nitrite) to volatile gases, nitrous oxide and/or molecular nitrogen. It is an enzymatic process accomplished by certain bacteria capable of using nitrate in place of oxygen as the terminal electron acceptor. Payne (1973) states that there are species in 15 genera of bacteria that have been reported to denitrify (32). These include Aggro- mobacter, Alcaligenes, Bacillus, Chromobacterium, Coryne- bacterium, Halobacterium, Hyphomicrobium, Micrococcus, Mor- axella, Nitrosomonas, Propionibacterium, Pseudomonas, Spirillum, Thiobacillus, and Xanthomonas. The most promin- ent denitrifying bacteria previously reported in soils were Bacillus spp., especially Bacillus cereus. In soils where nitrate fertilizers were added, these forms were replaced by Pseudomonas, Achromobacter, and Bacillus macerans (23). Denitrification was first recognized in 1886 by Gayon and Dupetit (19). They observed that certain soil bacteria were capable of reducing nitrate to molecular nitrogen and nitrous oxide. With the work bf Ferguson and Fred (1908), it was well established that denitrification in soils was enhanced by the addition of manure (16). Kluyver and Donker (1926) discovered that nitrate served as the hydro- gen acceptor in enzymatic dehydrogenation of organic or inorganic substrates (28). However, even with all that was known about denitrification at that time, scientists were not convinced that there was any economic or health significance to the process. Little interest was paid to the subject until 1946. It was then that the astronomer Adel observed that the concentration of nitrous oxide was greater near the surface of the earth than at higher a1- titudes. He suggested that this was due to a biological decomposition of nitrogen compounds in soil (2,3). Al- though the enhancement of denitrification is desired for the removal of nitrate from waste treatment systems and from groundwater, present concern centers around the detri- mental effects of the process. Denitrification losses of nitrogen from fertilizers added to soils have been re- ported to vary between 1 and 75% of that applied. However, many soil scientists feel that on the average 10 to 15% of the applied nitrogen is lost due to denitrification (9).- With the recent increased cost and world shortage of nitro- gen fertilizers, such a loss results in a financial loss to the farmer as well as reduced food production. Further- more, the denitrification intermediate, N 0, apparently 2 diffuses into the stratosphere and photo-decomposes to N2 and small amounts of NO and N02, which react with O to 3 form 02. Since ozone attentuates the UV light from the sun, such a depletion of O by N 0 means more UV light 3 2 will reach the Earth's surface and therefore pose a hazard to both plants and animals. Recently, McElroy has hypo- thesized that the increased global use of nitrogen fertil- izer has increased the amount of N O eminating from soil 2 thus enhancing ozone destruction. In 1974, almost 40 million metric tons of nitrogen fertilizers were used (11). There is little known about denitrification in natural ecosystems due to the absence of a sensitive, convenient, and specific assay for the process. Because of such tech- nical problems, one must rely more on ecological information obtained with pure cultures. However, one must have some indication of the importance of the culture in actual denitri- fication in the environment. Therefore, isolating and quant- ifying numerically dominant denitrifiers from a wide variety of habitats was a primary objective of this study. From this information, the degree of commonality of the genera and species with apparent ecological significance was de- termined. Secondary objectives included the development of an effective isolation and identification system for denitrifiers, the determinations of environmental factors which control population density and species composition, and the estimation of general population structure between organisms capable of anaerobic growth, those organisms which convert nitrate only to nitrite, and denitrifiers. MATERIALS AND METHODS Description Of Samples Nineteen soils, three fresh-water sediments, and a poultry manure liquor from a fermenter which was undergoing nitrification and denitrification were used. Samples were collected from locations where loss of nitrogen by denitri- fication had been measured or was expected. Soils were ob- tained from seven countries under a permit which allowed entry of untreated samples. The Connecticut soil, No. 1, was a sample obtained from an experimental soil column which was incubated with glucose and actively denitrifying (39). The Connecticut soil, No. 2, was from the same soil column after the com- pletion of the experiment,-. at which time denitrification was no longer being measured. Michigan Ponds l and 4 are two ponds of a four pond secondary effluent sewage treat- ment system known as the Water Quality Management Project presently in operation at Michigan State University. Pond l is the first pond in the system, thus receives the great- est organic load and Pond 4 is the last pond in the system, and has the lowest organic load. The pond retention times in the summer months are approximately 30 days. Ponds 1 and 4 were both sampled in November 1974 and May 1975. The nitrified poultry manure liquor was obtained from a nitrified mixed liquor, deoxygenated by bubbling N through 2 it to stimulate denitrification. The incubation tempera- ture of the liquor was 20 C (34). Contributors of the other samples (all soils) were asked to provide information on crop grown, previous crops, soil type, approximate location where sample was taken, and any other information thought to be useful. A summary of this information is listed in Table 2. Also, a listing of the contributors and their mailing addresses can be found in the Appendix. Contributors were asked to collect and ship the sample as described below. Sample Collection, Handling, And Storage Each composited soil sample of approximately 0.5 kg was made up from six subsamples freshly collected from the Ap (plow) horizon in a 10 m2 homogeneous soil area. The composited sample was then sealed in a plastic bag and placed in a sturdy container for immediate shipment by air. When received, each sample was refrigerated at l C until use. No sample was stored more than four weeks before use except for the Minnesota, California, Connecticut, and Kansas samples which were stored until the methods for the isolation and enumeration were standardized. The three sediments were collected with a plexiglass gravity corer. For Ponds 1 and 4, a composite sample was made from the top 1 cm of each core from three sites for each pond. The composite samples were stored at l C until use . Sample Characterization The soil classification is according to the 7th Approximation. For non-U.S. samples, the classification is only approximate. With the help of Dr. E. P. Whiteside, Michigan State University, and using FAO Soil Maps, infor- mation supplied by the cooperator, and the soil sample, an approximate equivalent for the 7th Approximation was deter- mined. Soil and sediment pH was obtained by mixing 15.0 g of a sample with 15.0 ml of distilled water. After allow- ing the suspension to settle for approximately 30 min, the mixture was again stirred and the pH reading taken on an Ionalyzer Model 801/Digital pH Meter (Orion Research Inc., Cambridge, Mass.). The pH meter was calibrated at 4.0, 7.0, and 10.0. A direct pH measurement was taken on the nitrified poultry manure sample. Soil conductivity was done according to the method of the U.S. Salinity Labor- atory Staff (6 ). Soil texture measurements were done according to the manual ribbon method by Dr. D. Mokma, Michigan State University, and by the hygrometer (appendix data only) method. Organic matter compositioncxfthe samples was done by the wet combustion method of Allison ( 7). Approximately 10 g of soil was used for percent moisture. determinations. Soils were dried overnight at 110 C and then weighed. Mean annual precipitation and mean annual temperature of the sample sites were obtained from maps (20,24). Isolation And Enumeration After preliminary testing of media and procedures for the recovery of bacteria from soil had yielded a satisfac- tory procedure, denitrifiers were isolated and enumerated according to the scheme shown in Figure l. The first dilution was prepared by blending 10.0 g of sample in a Waring blender for 2 min with 90 ml distilled water and a final concentration of 0.1% Tween 80. Isolations were accomplished by spread plating 1.0 ml inocula of appropri- ate dilutions onto nitrate agar. The agar plates had been pre-dried for 3 to 5 days, thus allowing rapid absorbtion of the inocula. Two dilution series were done for each sample and four plates were prepared for each dilutionin 4 to 10.6 range, unless unusual numbers were antic- the 10- ipated. The plates were then incubated for 3 to 5 days at room temperature in an anaerobic glove box (4), with 90% as the atmosphere. After incubation, all of N 10% H 2' 2 the isolated colonies (approximately 15 to 60) from at least one plate of each series were transferred to nitrate broth (Difco, Detroit, Mi-) tubes containing an inverted (Durham) tube and incubated anaerobically for two weeks. In addition, colonies of different morphology from other plates were also transferred. Gas producers were purified on nitrate agar incubated anaerobically and then transferred 10.0 g of soil blended for 2 min in 90.0 ml of water with 0.1 % Tween 80; dilutions made Nitrate broth tubes inoculated with appro- priate dilutions; tubes incubated for 2 weeks. Broth assayed for N03 and N02; MPN determined Gas produEggg—noted; broth of non-gas pro- ducers tested for N03, N02, and NH4 Stocks made OI purified gas producers _NH 1.0 m1 inocula of appro- priate dilutions spread plated on pre-dried ni- trate agar; plates incu- bated for 3 to 5 days Colonies picked and trans- ferred to Durham nitrate broth tubes; incubated for 2 wee Stocks made of colonies Gas producers streaked for purit Nitrate broth, with Durham inserts with septa, inocu- lated with purified gas producers; incubated for 2 weeks Gas producers analyzed for N2, N20, and C02; broth tested for N03, N02, and 4 Gas producers confirmed as denitrifiers Figure 1. Scheme Utilized for MPN, Isolation, and Confirma- tion of Denitrifying Bacteria.1 1All incubations were in an anaerobic glove box, except stocks made of pure gas producers were incubated aerobically. again to nitrate broth tubes but containing Durham tubes with septa on the upper end. After a two or more week anaerobic incubation, the broth of those that did not produce gas was assayed qualitatively for nitrate, nitrite, andammonia. Gas produced by the purified isolates was analyzed for N2, N20,and CO2 by gas chromatography. Con- firmed denitrifiers were those organisms able to pro- duce N2 and/or N20 after purification. The method of Focht and Joseph was used for MPN de- terminations for denitrifiers (18). The method was mod- ified by utilizing an anaerobic glove box for incubations. Five tubes of nitrate broth per dilution were used. In 5 to 10-8. For each most cases, the dilution range was 10- sample the same two dilution series described above were used to inoculate the MPN broth tubes. After 14 days incubation, the broth of each tube was observed for tur- bidity and assayed qualitatively for nitrate and nitrite. Tubes in which no nitrate or nitrite was detected4were positive for the presence of a denitrifying organism. Tubes in which nitrite was found were positive for the presence of a nitrite accumulating organism. Turbidity indicated the presence of an anaerobe. (In this thesis "anaerobe" is defined as any organism capable of growth under anaerobic incubations.) For the soils and sediments, MPN's and plate counts are expressed as mean organisms per gram dry weight. The population for the nitrified poultry manure is reported as organisms per ml. 10 Methods Nitrate and nitrite were determined qualitatively according to the method of Focht and Joseph (18). Quan- titative measurements for nitrate were done potentiometric- ally with a Ionalyzer Model 801/ digital pH meter equipped with a nitrate ion electrode (Orion Research Inc., Cam- bridge, Mass.). Quantitative measurements for nitrite were done by the Griess-Ilosvay method (7) using a Turner Spec- trophotometer Model 350 (G.K. Turner Associates, Palo Alto, Cal.) for the colorimetric readings. Ammonia was detected qualitatively with Nessler's reagent (37). Nitrogen, nitrous oxide, and carbon dioxide were quantitated using a Carle Model 8000 gas chromatograph (Carle Instruments, Inc., Fullerton, Cal.) equipped with poropak Q and molecular sieve 5A columns and a micro— thermister detector. The limit for detection of N20 was 0.5 % of a 50 ul sample. All anaerobic incubations were done at room temperature in an atmosphere of 90% N2: 10% H2 contained in a vinyl glove box (Coy Manufacturing, Ann Arbor, Michigan). The methods used in setting up the glove box were those of Aranki et a1. (4). The atmosphere of the glove box was sufficient to maintain reduced resazurin, (Eh < -50 mV). The modified Durham inserts consisted of 7.6 cm pieces of glass tubing with an inner diameter of 5 mm. A serum septum (Arthur H. Thomas Company, Philadel- phia, Pa.) in one end allowed for the insertion of a 25 gauge needle. Thus, gas collected could be removed and ll assayed by gas chromatography. Denitrifier Characterizations The confirmed denitrifier isolates were characterized by examining the 52 properties identified in Table 1. A11 cellular characteristics were examined on early exponential phase cells grown aerobically on nitrate broth or agar at 28 C, unless otherwise indicated. Gram stains were done according to the Kopeloff modification (25). Cellular morphology, cell groupings, and motility were determined by hanging drop observations (1). Cells were measured under phase contrast microscopy after mounting on slides containing a thin film of dried water-agar (12). To ob- tain the range of dimensions, the smallest and largest cells of several fields were measured. For poly-B-hydrox- ybutyrate formation, cells were grown on Difco nutrient agar supplimented with 2.0 % glycerol and 1.0 % glucose (filter-sterilized separately). Cells were stained by Burdon's method (13). Colonial characteristics of size, form, elevation, margin, surface, texture, and light refraction and catalase and oxidase reactions were all observed on 3 day-old cul- tures grown aerobically on nitrate agar at 28 C (13). Taxo Differentiation Discs for Neisseria and Pseudomonas (BBL, Division of Becton, Dickinson and Company, Cockeysville, Maryland) were used for the oxidase test. The presence of arginine dihydrolase was determined 12 Table 1” Characters Examined for Each Denitrifier Isolate. II. III. IV. VI. Cellular characteristics: VII. gram stain morphology cell groupings motility PHB inclusions cell length cell width Colonial characteristics: size form elevation margin surface texture light refraction Enzyme ppoduction: catalase oxidase oxidation of arsenite arginine dihydrolase Hydrolytic capabilities: gelatin starch casein Temperature for growth: 4 C 28 C 41 C Pigment production: fluorescein (UV) general pigment (diffusible and non-diffusible) insoluble blue phenazine pigment Growth as sole carbon source: acids acetate propionate citrate p-hydroxybenzoate alcohols ethanol geraniol amino acids L-asparagine DL-arginine B-alanine sarcosine Carbohydrates and sugar derivatives D-glucose sucrose D(+)trehalose L-arabinose D-fructose D-arabinose D-xylose D-ribose maltose D(+)cellobiose 2-keto gluconate saccharate Polyalcohols and glycols D-sorbitol meso-inositol propylene glycol 13 by the method of Thornley (41). Two tubes of Thornley's medium "2A" were inoculated for each isolate. One of the two tubes was incubated aerobically at 28 C for 4 days. The method was modified by incubating the other tube in an an- aerobic glove box, instead of covering with vaseline, at room temperature for 4 days. An alkaline reaction in both tubes was positive for arginine dihydrolase. The ability to oxidize arsenite was determined by the method of Turner (42), after incubating the medium aerobically at 28 C for one week. The ability to grow at 4 C and 41 C was determined by turbidity after aerobic incubation in nitrate broth. The incubation period for 4 C was 10 days, while that of 41 C was 2 days. Tests for hydrolytic capabilities, pigment production, and growth on sole carbon sources were performed after aerobic incubations at 28 C for 4 days. Geraniol medium was incubated 7 days. The media and methods of analysis used to determine the hydrolytic capabilities for starch was that of Colwell and Wiebe (13); for gelatin, Frazier (30); and for Casein, Gordon and Mihm (21). The ability to produce three types of pigment was examined. Medium B (27) was used for the enhancement of fluorescein production. Medium A (27) was used for des- cription of general diffusible and non-diffusible non- fluorescent pigment production. A peptone-glucose medium 14 (38) was used for the enhancement of the production of an insoluble blue phenazine pigment characteristically pro- duced by Pseudomonas fluorescens biotype IV. For Medium A and the peptone-glucose medium, pigments were examined under white light. For Medium B, plates were examined under long wave ultraviolet light. Every isolate was tested for the ability to grow at the expense of 25 different organic compounds. Thetest media were prepared by adding each organic compound, at the appropriate concentration, to the standard mineral base of Colwell and Wiebe (13), with the addition of .001 % phenol red (Sigma Chemical Co., St. Louis, Mo.). The pH indicator aided in evaluation of growth since the metabOl- ism of most of the compounds produced a pH change. The final pH of all media were 7.2. Oxoid Ionagar no. 2 (Colab laboratories, Inc., Chicago Heights, Ill.) was used as the solidifying agent. The media were contained in Quad petri dishes. A control plate, without an added organic compound, was inoculated with each isolate. Growth on plates with the carbon sources compared to the control plates was read as positive for the ability to utilize the sole carbon source. Geraniol media was prepared by adding a drop of the water-insoluble geraniol to 10.0 ml of the mineral base of Tiedje and Mason (40), and adding the phosphate buffer which had been autoclaved separately. Turbidity in excess of the uninoculated control was con- sidered positive for the ability to utilize geraniol as a 15 sole carbon source. The organic compounds tested as sub- strates and the concentrations used were:1 (a) Acids: 0.1 % acetate (A), 0.1 % propionate (A), 0.1 % citrate (B). (b) Alcohols: 1.0 % ethanol (G), geraniol (C). (c) Amino acids: 0.1 % L-asparagine (D), 0.1 % DL- arginine (D), 0.1 % B-alanine (D). (d) Carbohydrates and sugar derivatives: 1.0 % D- glucose (B), 1.0 % sucrose (B), 1.0 % D(+)trehal- ose (D), 1.0 % L-arabinose (D), 1.0 % D-fructose (D), 1.0 % D—arabinose (D), 1.0 % D-xylose (D), 1.0 % D-ribose (D), 0.1 % maltose (F), 0.1 % D(+)cellobiose (D), 0.1 % 2-keto gluconate (D), 0.1 % saccharate (D). (e) Polyalcohols and glycols: 1.0 % D-sorbitol (D), 1.0 % meso-inositol (D), 0.1 % propylene glycol (D). (f) Miscellaneous: 0.1 % p-hydroxybenzoate (C), 0.1 % sarcosine (E). The following organic compounds were filter-sterilized: ethanol, D—glucose, sucrose, D(+)trehalose, L-arabinose, D-fructose, D-arabinose, D-xylose, D-ribose, maltose, 1The following designations are used to indicate the source of the sole carbon sources: A) J.T. Baker Chemical Com, Phillipsburg, N.J.; B) Mallinckrodt Chemical Works, St. Louis, Mo.; C) Aldrich Chemical Co., Inc., Milwaukee, Wis.; D) Sigma Chemical Co., St. Louis, Mo.; E) Columbia Organic Chemical Co., Inc., Columbia, S.C.; F) Difco, Detroit, Mi.; G) Commercial Solvents Corporation, Terre Haute, Ind. 16 D(+)cellobiose, 2—keto gluconate, D-sorbitol, and meso- inositol. In addition to the characterization of the isolated denitrifiers, the same characters were examined for nine known denitrifiers. They were: Pseudomonas denitrificans ATCC 13867, Pseudomonas aureofaciens ATCC 13985, Pseudo- monas mendocino ATCC 25411, Alcaligenes faecalis ATCC 8750, Pseudomonas fluorescens II ATCC 17822, Pseudomonas aerugin- osa, Paracoccus denitrificans ATCC 2008, Pseudomonas stut- zeri ATCC 17588, and Pseudomonas perfectomarinus from Spartina salt marsh (isolated by W.J. Payne). Effect Of Phosphate In Media Nitrate broth, autoclaved with the molar concentra- tions of 0.02, 0.015, 0.01, 0.005, and 0.0 phosphate buffer, pH 7.2, was tested for the ability to support growth and gas production of selected sediment isolates. Twenty-one isolates were obtained from Pond 1 in November. Durham broth tubes containing the five phosphate concentrations were inoculated.with each of the 21 isolates. After a two week anaerobic incubation, turbidity and the gas volumes in the Durham tubes were recorded. The effect of filter-sterilized phosphate buffer on the growth and gas production of known denitrifiers was also observed. Nitrate broth tubes with Durham inserts with septa were prepared with final molarities of 0.025, 0.02, 0.015, 0.01, 0.005, and 0.0 phosphate buffer. A tube of each of 17 the five phosphate buffer concentrations was inoculated with each of the ten confirmed or known denitrifying bac- teria. The denitrifiers were Pseudomonas fluorescens (DMS l9), Pseudomonas perfectomarinus, Hyphomicrobium sp. ‘(WC 24 R), Pseudomonas dentrificans (ATCC 13867), Paraco- ccus denitrificans (ATCC 2008), Pseudomonas stutzeri (ATCC 17588), Alcaligenes eutrophus,1 and isolates 4, 15, and 49 (confirmed denitrifiers by the author). After a terminal incubation period, the volume of gas produced in each tube was measured. The gas was analyzed for N2 and N20 by gas chromatography. Effect Of Tween 80 Tween 80 has been used by other workers (36) to aid in the recovery from soil of bacteria imbedded in organic matter films. To test the efficacy of this method, a com- parison test was done using three concentrations of Tween 80 SC-15608 (Sargeant-Welch Scientific Co., Skokie, Ill.). Ten grams of Minnesota soil were blended in a Waring blender for 2 min with 90.0 ml distilled water with a final concentration of either 0.1%, 0.05 %, or no Tween 80. Anti- foam A spray (Dow Corning Corp., Midland, Mi.) was used after blending. Nitrate broth tubes autoclaved with 0.02 M phosphate buffer, pH 7.2, were inoculated with appropriate 1The named denitrifiers were from the collection of W.J Payne. The ATCC numbers were the original numbers and not directly obtained by us from the American Type Culture Col- lection. Pseudomonas fluorescens was originally from Dr. Clarke Gray, Dartmouth Medical School. Hyphomicrobium sp. was originally from Dr. G.T. Sperl. The Alcaligenes eutro- phus (strain H 16) was originally from Dr. H. Kaltwasser. 18 dilutions. After a two week anaerobic incubation, the broth of each tube was qualitatively assayed for nitrate and nitrite to obtain the MPN of denitrifiers. gomparison Of Nitrate Broth With Soil Extract-Yeast Extract 1.35221; A Several media were employed to determine which would give the greatest numbers of denitrifying bacteria. Soil extract was prepared according to the method of Lochhead (26). The source of the soil extract was a Brookston loam with an organic matter composition of 3.4 %. After the soil-water mixture was autoclaved and allowed to settle, the supernatant was removed. Lochhead's method was modi- fied by centrifuging (instead of filtering) the supernatant to remove the clay particles. The resulting clear soil ex- tract was used immediately for media preparation. A soil extract broth was prepared by supplementing the soil ex- tract with 0.1 % yeast extract and 0.1 % KNO , buffered 3 with potassium phosphate to pH 7.2. The soil extract broth and nitrate broth with 0.02 M potassium phosphate buffer, pH 7.2, were compared for highest MPN's of denitrifying bacteria. The soil used for the inoculations was the Minn- esota. RESULTS Sample Information And Physical Measurements Table 2 summarizes information on the samples. The sample range included temperate agricultural, sub-tropical, tropical, rain forest, rice paddy, desert, waste treated soils, a nitrified poultry manure, and freshwater lake sediments. Major soil groups represented included molli- sol, histisol, vertisol, entisol, inceptisol, aridisol, and alfisol. Most of the soils did not have crops at sampling time, but those that did had either wheat, corn, or rice. A wide range of pH values (3.8 to 8.2) was represented. Most of the samples had 1 to 5 % organic matter, which is typical of mineral soils. The Venezuelan soil was unusually high in organic matter because of the generally high water table which often reached the surface. Soil texture ranged from very heavy clay soils, eg. vertisols, to very sandy soils. An estimate of the actual percentages of sand, silt, and clay, as determined by the hygrometer method, can be found in Appendix. Medium Selection When comparing MPN's of denitrifiers obtained from the Minnesota soil, the nitrate broth autoclaved with 0.02 M phosphate buffer yielded a population estimate (1.66 x 106 19 20 Table 2. Summary of Information on the Samples Provided by Contributors or Determined Experimentally. Sample} Sagpting General Description 1 Minnesota Lamberton' Agric soil 12" - 18" 2 California Davis Agric soil 3 Connecticut 1 Windsor Agric soil 4 Connecticut 2 Windsor Agric soil 5' Argentina (SP) San Pedro Agric soil 6 Argentina (B) Balcarce Agric soil 7 Michigan (muck) Bath Organic agric soil 8 Texas Temple Agric soil 9 Argentina (P) Parana Agric soil 10 Brazil Mococa Agric soil 11 Venezuela San Carlos de Rain forest Rio Negro 12 Nigeria (C) Ibadan Agric soil 13 Nigeria (R) Ibadan Rice paddy 14 Columbia Palmira Rice paddy 15 Philippines Los Banos Rice paddy 16 Taiwan Taichung Rice paddy 17 Louisiana Crowley Rice paddy 18 Utah Snowville Desert 19 Kansas Pratt Manured agric soil 20 Poultry waste - Poultry waste 21 Michigan (WG) Hickory Wintergreen Lake Corners sediment 22 Michigan (Pl) E. Lansing WQMP sediment, pond 1 23 E. Lansing WQMP sediment, Michigan (P4) pond 4 21 Table 2. (continued) Classification Crop at Samp- Previous Drainage1 (series) ling Time Crops 1 Typic haplaquoll - - p Webster 2 Typic pelloxerert - - 9 Clear Lake 3 Entic haplorthod none tobacco w Merrimac 4 Entic haplorthod - — w Merrimac 5 Vertic argiudoll fallow sweet corn swp Ramallo 6 Mollisol ' none (plowed potato w Typic argiudoll after wheat) 7 Histisol-Typic - - - 7 medisaprist(Car1isle) 8 Typic chromudert - - P Houston 9 Argillic chromudert fallow wheat swp Febre 10 Entisol wheat rice p Tropic fluvaquent 11 Inceptisol - - P Tropaquept 12 Agric hapustalf corn corn swp l3 Agric hapustalf rice corn, rice p 14 Inceptisol rice rice swp Andaquept 15 none rice swp 16 Entisol rice rice swp/p Fluvaquent 17 Al f isol-Thermic typ- none rice swp ic a1baqualf(Crowley) l8 Calcorthid Artemisia none w Thiocal ' tridentata 19 Mollisol corn corn - Aquent 20 - - - - 21 Hypereutropic — — - 22 - - - - 23 - — - - 1Drainage designations as follows: w - well, p - poor, swp - somewhat poorly. 22 Table 2. (continued) Nitrogen Fertilizer Use Moisture on Organichatter in the Last Two Years receipt (%) pH ' ”(%)‘ ’* l - 15.9 7.2 2.34 2 - 20.8 7.34 2.16 3' - 8.7 5.2 1.45 4 - 10.75 5.2 1.45 5 none 22.3 5.92 3.34 6 unknown 23.0 5.69 4.08 7 - 84.5 6.53 81.51 8 - 6.55 7.39 3.61 9 no 28.6 7.84 2.81 10 80 kg N/ha 24.7 4.42 4.94 11 - 56.4 3.84 16.15 _12 yes 12.75 5.51 3.21 13 yes 17.2 6.34 1.34 14 '125 kg urea/ha .32.0 7.74 3.21 15 no 31.0 6.49 3.34 16 2200 kg (NH4)ZSO4/ha 19.6 5.09 1.20 17 unknown 17.5 5.66 1.07 18 no 3.7 8.21 2.34 19 320 T/ha/year 10.9 7.12 4.81 animal waste 20 - - 6.44 46.00 21 - 92.0 6.41 26.47 22 - 33.8 - - 23 - 35.0 6.94 2.50 23 Table 2. (continued) Texture 5.23333” ”T‘Zigei‘ii‘fii‘i ”322.221?“ Reference 11 mhos (C) (mm) 1 cl 334 5-10 500-1000 (31) 2~ sic 618 15-20 400-500 3 $1 350 10 1000 (39) 4 31 350 10 1000 (39) 5 sicl 433 15-20 500-1000 6 sil 687 15-20 500-1000 7. muck 225 5-10 .500-1000 8 c 2146 20-25' 1000-2000 9 c 315 15-20 500-1000 10 sicl 303 20-25 1000-2000 11 1 241 25-30 2000-4000 12 sl 629 25-30 1000-2000 13 ls 238 25-30- 1000-2000 14 sicl 398 25-30 1000-2000 15 sicl 386 25-30 1000-2000 16 sil 995 20-25 1000-2000 17 sil 521 20-25 1000-2000 18 sil 705 10-15 250-500 19 sil 3623 10-15 500-1000 (43) 20 - - - - (34) 21 - 579 5-10 500-1000 22 - - 5—10 500-1000 23 - 1031 5-10 500-1000 24 org/g dry wt) similar to that of the soil extract broth supplimented with 0.1% yeast extract and 0.1 % KNO (1.12 3 x 106 org/g dry wt). This suggests that nitrate broth did not lack any growth factors essential for the growth of soil denitrifiers. Since the population estimates were approximately the same and the preparation of nitrate broth less time consuming, all subsequent experiments were done with nitrate broth or agar. Effect Of Tween 80 As A Soil Dispersing Agent When using 0.1 % (v/v) Tween 80 as a soil dispersing agent with the Minnesota soil, a mean MPN of 6.07 x 105 org/g dry wt for two dilution series was obtained. The values for 0.05 e and 0.0 % were 2.54 x 105 org/g dry wt and 1.95 x 105 org/g dry wt, respectively. Use of Tween 80 did not appear to have a bacteriocidal effect. Since the three values were within an order of magnitude, I cannot be confident whether Tween 80 aided in the recovery or had a toxic effect on soil organisms. However, it was 'apparent that the use of Tween 80 with the Waring blender greatly facilitated the mixing of soils high in clay con- tent. For such soils, foaming did occur, but was reduced by the addition of antifoam A. A concentration of 0.1 % Tween 80 was used for all soil preparations. Effect Of Phosphate Table 3 summarizes the effect that autoclaved phos- phate buffer has on growth and gas production of Pond 1 25 Table 3. The Effect of Phosphate Buffer on the Growth and Gas Production of Selected Soil and Sediment Organisms. Phosphate Isolates Isolates Isolates Mean Gas Concentra- Trans- Producing‘ Producing Volume per tion (M) ferred Turbidity Gas Producer (pl) autoclaved 0.000 21a 14 11 95 0.005 21 11 7 78 0.010 21 9 9 48 0.015 21 9 8 36 0.020 21 2 1 trace filter-sterilized 0.000 10b 10 10 133 0.005 10 10 10 128 0.010 10 10 9 146 0.015 10 10 9 157 0.020 10 10 9 155 0.025 10 10 8 145 a21 isolates from Pond 1 (November) sediment. b7 identified and 3 unidentified cultures of denit- rifying bacteria. They were: Pseudomonas fluorescens (DMS 19), Pseudomonas perfectomarinus, Hyphomicrobium (WC 24 R), Pseudomonas dentrificans (ATCC 13867), Paracoccus denitrificans (ATCC 2008), Pseudomonas stutzeri (ATCC 17588), Alcaligenes eutrophus, and isolates 4, 15, and 49. (November) sediment organisms. Although the organisms were not identified or confirmed as denitrifiers, the data 26 clearly shows that the presence of autoclaved phosphate buffer is inhibitory to the growth and gas production of the 21 sediment organisms. The effect of filter-sterilized phosphate buffer on growth and gas production can be seen in Table 3. All ten of the stock denitrifiers grew in all of the phos- phate concentrations. However, only in .000 and .005 M buffer did all ten produce gas. The higher concentrations of buffer did affect the gas production of specific organ- isms. The average volume of gas produced varied insig- and traces of CO and 2 2 N20. In the broth of all 10 stock denitrifiers, for all of the phosphate molarities, no nitrate was detected. nificantly, with 99 % of the gas N Nitrite was detected in trace quantities in the broth of Alcaligenes eutrophus at 0.01 M, 0.015 M, and 0.025 M buffer; of Hyphomicrobium at 0.02 M buffer; of Pseudomonas stutzeri at 0.015 M buffer; and of isolate 15 at 0.025 M buffer. Alcaligenes eutrgphus did not produce gas in broth of phosphate concentrations greater than .005 M and isolate 4 did not produce gas at 0.025 M. Although the effect of filter-sterilized phosphate buffer was not as pronounced as that of the autoclaved phosphate buffer, it was evident that both treatments exhibited an inhibitory effect on the growth and gas pro- duction of certain soil organisms. The media chosen for use in isolating and enumerating denitrifying bacteria was unbuffered nitrate broth and agar. 27 Quantitation 0f Total Anaerobes, Denitrifiers, And Nitrite Accumulators Table 4 lists the mean population estimates of total anaerobes, denitrifiers, and nitrite accumulators using both MPN and plate count methods. With few exceptions, the MPN and plate counts were in good agreement. Of the 25 samples done for MPchfdenitrifiers, 72% of the values fell in the range of 105 to 107 organism3°g-l. The excep- tions which fell below 105 organisms-g"l were samples from Pond 4 sediment (November), Utah, Venezuela, and Texas. The Pond l sediment (November)J nitrified poultry manure, and Connecticut 1 samples had values above 107 organisms-g- or °m1-l. The MPN values of nitrite accumulators were greater than those of the denitrifiers. Of the 22 samples examined for nitrite accumulators, 59 % of the MPN's were 6 7 6 and 10 between 10 organisms-g.l and 91 % ranged from 10 to 108. Only the Venezuelan soil had an MPN of less than 106 nitrite accumulators per gram dry weight. For the 25 samples examined for MPN of total organisms capable of growth under anaerobic incubations, 76 % were in the range 6 8 of 10 to 10 organisms-9-1. An outstanding exception was more than 1010 organisms~ml-l for the nitrified poultry manure. In some cases, nitrite accumulator numbers were read to be higher than those of total anaerobes. This was presumably due to the greater sensitivity of the nitrite detection method compared to visual detection of turbidity. Table 4 also illustrates numerical relationships of 28 Table 4. Population Densities and Ratios of Denitrifiers, Nitrite Accumulators, and Total Anaerobes. Denitrifiersa Nitrite Accumulators Sample MPN Plate MPN Plate 1 Minnesota 2.88x106 1.38x106 6.48x106 1.38x106 2 California 2.38x106 2.08xlo6 1.58x107 2.92x106 3 Connecticut 1 1.37x107 1.08x106 1.37x107 2.98x106 4 Connecticut 2 1.42x105 l.10x105 2.00x105 19 Kansas 2.28x105 2.22x105 1.04x107 1.00x106 20 poultry waste 3.50x1010 2.00x108 - 1.38x108 aNumbers expressed as organisms-g waste which is expressd as organisms-ml‘ . except for poultry 29 Table 4. (continued) Denitrifiers Nitrite Accumulators Sample MPN Plate MPN Plate . . 5 5 6 ' 6 21 Michigan(wc) 3.44x10 3.75x10 1.17x10 1.56x10 22 Michigan(P1) 3.45x107 2.05x107 - - ¥°V9mber 6 6 7 6 23 Michigan(Pl) 3.70x10 l.8lx10 1.19x10 1.43x10 24 gay. 4 5 4 Michigan(P4) 5.23x10 1.92x10 - 7.68x10 November ‘ . 25 Michigan(P4) 3.77x106 7.55x105 3.16x106 7.55x105 May Anaerobes b b (MPN)a N : A D : A D : N 1 6.36x106 1.00~: 1° .44 : 1° .44 : 1 2 3.69x107 .43 : 1 .06 : 1 .15 : 1 3 >1.37x107 <1.00 : 1 <1.00 : 1 1.00 : 1 4 7.10x106 .18 : 1 .02 : 1 .11 : 1 5 1.01x107 .75 : 1 .14 : 1 .18 : 1 6 8.15x106 1.00 : 1° .03 : 1° .03 : 1 7 1.83x107 .79 : 1 .38 : 1 .49 : l 8 7.81x105 1.00 : 1° .009 : 1° .009 : 1 9 8.25x106 .31 : 1 .19 : 1 .62 : 1 10 -3.22x106 1.00 : 1° .15 : 1° .15 : 1 11 1.35x106 .63 : 1 <.03 : 1 <.05 : l b I u I N-nltrlte accumulators, A-anaerobes, D-dentrifiers c N counts used as A counts because of greater number d D counts used as A counts because of greater number 30 Table 4. (continued) M75161??? N : Ab D : Ab D : Nb 12 4.71x105 1.00 : 1° .09 : 1° .09 : 1 13 1.79x106 .83 : 1 .33 : 1 .39 : 1 14 6.78x106 .54 : 1 .04 : 1 .07 : 1 15 8.32x106 1.00 : 1° .07 : 1° .07 : 1 l6 1.13x107 1.00 : 1° .03 : 1° .03 : 1 17 6.52x106 1.00 :-1° .23 : 1° .23 : 1 18 >1.30x105 >.85 : 1 <.006 : l .007 : 1 19 1.20x107 .87 : 1 .02 : 1 .02 : 1 20 4.45x1010 - .79 : 1 - 21 2.88x106 .41 : 1 .12 : 1 .29 : 1 22 1.11x108 - .31 : 1 - 23 1.43x107 .83 : 1 .26 : 1 .31 : 1 24 3.53x105 - .15 : 1 - 25 2.00x106 .84 : 1° 1.00 : 1° 1.19 : 1° nitrite accumulators and denitrifiers to anaerobes and denitrifiers to nitrite accumulators. In most cases, nitrite accumulators were within an order of magnitude of total anaerobes. In some instances, nitrite accumulator values were greater than those of anaerobes. Since this is not possible, when calculating ratios in these instances, the nitrite accumulator numbers were used as total anaerobe numbers. Denitrifier numbers were generally within an 31 order of magnitude of both the anaerobe and nitrite accum- ulator counts. The Utah soil had the lowest number of den- itrifiers relative to nitrite accumulators of all of the soils - .007 : 1. Whereas, Pond 4 sediment (May) had a greater than 1 : 1 ratio of denitrifiers to nitrite accum- ulators. Excluding the Utah and Venezuela soils because of their extreme pH and the poultry manure sample and Connecticut soils because of their laboratory incubations, a mean population relationship for denitrifiers to nitrite accumulators to anaerobes of soils and sediments was cal- culated to be 0.24 : 0.68 : 1. The Ability Of Isolates To Utilize Nitrate From the 25 samples, 1553 isolates were tested for denitrification and 16.2 % of these were gas producers (Table 5). For each sample, the range for isolates pro- ducing gas was from 0.0 % to 50.0 %. Samples from which no gas producers were isolated were ConnectiCut No. 2, Utah, and the Venezuela soils. Pond 1 sediment (November) and Minnesota soil had the highest percentages of gas 'producers. The Pond 1 sediment (May) was next with 39.1 % of its isolates producing gas. Those organisms able to reduce nitrate to nitrite comprised 38.5 % of the 1531 isolates from 24 samples. The range of those reducing nitrate to nitrite was 4.5 % for the Pond 4 sediment (November) to 71.8 % for the Argentina(B) soil. After observing that the spent broth of certain 32 Table 5. Percent of Isolates Utilizing Nitrate and Pro- ducing the Following Nitrogen Compounds. $3322. 1 Minnesota 87 40.2 29.9 16.1 20.7 2.3 2 California 75 12.0 17.3 12.0 65.3 1.3 3 Connecticut 1 81 7.4 14.8 - - e 4 Connecticut 2 73 0.0 35.6 26.0 58.9 1.4 5 Argentina(SP) 87 26.4 32.2 24.1 31.0 . 6 Argentina(B) 39 2.6 71.8 41.0 20.5 . 7 Michigan(muck) 84 9.5 61.9 8.3 25.0 . 8 Texas 84 2.4 22.6 5.9 75.0 . 9_Argentina(P) 69 15.9 27.5 11.6 43.5 . 10 Brazil 54 20.4 37.0 31.5 22.2 . 11 Venezuela 45 0.0 33.3 26.7 66.7 . 12 Nigeria(C) 79 12.7 43.0 25.3 43.0 . 13 Nigeria(R) 48 18.8 50.0 45.8 27.1 14 Columbia 41 17.1 58.1 36.6 19.5 . 15 Philippines 124 9.8 30.6 41.1 25.8 . 16 Taiwan 40 15.0 50.0 37.5 35.0 . 17 Louisiana 42 16.7 47.6 23.8 42.9 . 18 Utah 66 0.0 69.7 10.6 30.3 . 19 Kansas 22 9.1 40.9 31.8 50.0 . 20 Poultry waste 67 34.3 17.9 - - - 21 Michigan(WG) 67 8.9 40.3 14.9 47.8 0.0 22 Michigan(Pl) 22 50.0 - - - - November 23 Michigan(Pl) 69 39.1 33.3 11.6 18.8 7.2 May . 24 Michigan(P4) 66 9.1 4.5 - - - November 25 Michigan(P4) 22 27.3 54.5 40.9 27.3 0.0 May isolates did not contain nitrate or gas, assays were done for ammonia. nitrite, or produced From 21 samples with a 33 total of 1317 isolates, 24.9 % showed increased ammonia in the medium.. For the individual samples, the range was from 5.9 % for the Texas sample to 45.8 % for the Nigeria(R) soil. Even after assaying for ammonia, certain isolates were discovered that were able to deplete the broth of nitrate without subsequent gas, nitrite, or ammonia for- mation. Although their numbers were few, they did repre- sent 1.6 % of 1317 isolates tested. From the 21 soils, they were found in 9. The Argentine(P) soil had the high- est percentage of this group with 8.7 %. The next most prevalent group of isolates were those unable to utilize nitrate. That is, nitrate was detected without the formation of gas, nitrite, or ammonia. These isolates represented 37.9 % of 1317 tested from 21 samples and ranged from 18.8 % of the isolates for Pond 1 sediment (May) to 75.0 % for the Texas soil. Many of these organisms did not appear to grow in the nitrate broth after transfer from agar. Identification Of Isolated Denitrifying Bacteria Over 250 organisms were isolated that initially pro- duced gas (Table 5). However, many of these isolates were no longer viable after short anaerobic incubation periods or else lost the ability to produce gas after purification. Of the original gas producers, 147 denitrified after pur- ification; they were isolated from 19 of the 25 samples. These 147 confirmed denitrifiers,as well as nine stock 34 cultures were characterized by examining 52 properties for each. The results are recorded in the Appendix. Many of the organisms were identified to the species level by the current taxonomic criteria of Bergey's Manual of Determina- tive Bacteriology, 8th edition (10), appropriate supplimen- tary literature (38, 14, 33)), and comparison to known denitrifiers. Approximately one-third of the isolates were not identified to the species level and several were not identified to the genus level. Such isolates which appeared to be related based on the 52 properties tested, were given type numbers in the order in which they were isolated. Figure 2 illustrates a simplified characterization scheme used for the identification of the denitrifiers. Table 6 lists the identity of the organisms and their numbers for each sample. Gram negative rods comprised 93.2% of the 147 den- itrifiers and gram negative motile rods, mostly oxidase and catalase positive, represented 86.4% of the total. Other groups were: gram variable rods, 4.8 % pleomorphic strains 1.4 %; and gram positive cocci, < 1.0 %. Some samples were represented by a single species, such as the nitrified poultry manure, and the Brazilian, Kansan, and Michigan (muck) soils. However, from most soils several species were isolated. The greatest diversity was found in the Argentina (SP) soil where 10 different species were recovered. The species composition and number of species 35 rod or coccobacillus “‘?___——r. I‘a.=::---i;:f‘-——.._‘i-L Gram - l‘rlijELIL“‘::§r + motility - motility + acetate — spores + oxidase + violet pigment + catalase + yellow non- . . diffusible " Chromobzcterlum Bacillus pigment ace te p. p. Flavobacterlum sp. perltrlchous polar flagella flagella I Alcaligenes SP- Pseudomonas sp. glucose - glucose + starch + fructose - fructose + gelatin - propionate + geraniol - growth @ 41 + A. faecalis A. eutrophus P. s!utzeri 5“ gelatin + starch - . gelatin _ (continued on next page) growth @ 41 + growth @ 41 - \> glucose - sucrose + etha l + D-arabinose + D-arabinose - glucose + Sf YG diffusible geraniol + thanol - P. denltrificans non-flourescent ATCC 19244 pigment sucpose + sucrose - P. solanacearum P. caryophylli P. mendocino P. denitrificans ATCC 13867 Figure 2. Characterization Scheme for the Identification of Soil and Sediment Denitrifiers. 36 Figure 2. (continued) starch + starch d1 growth @ 41 + growth @ 41 + D-xylose - D-xylose + D-ri_ se + D-ribose - P. pseudomallei P. mallei trehalose - geraniol + growth @ 41 + P. aeruginosa non-diffusible non-fluorescent green pigment ethanol + orange-yellow non-fluorescent pigment P. aureofaciens P. chlororaphis P. L-arabinose + sucrose - starch - UV fluorescent pigment on King medium E trehalose + geraniol - growth @ 41 - L-arabin- ose + sucrose + fluorescens III propionate + propylene glycol + ethanol + P. fluorescens II 1 all strains studied (10). propionate - propylene glycol - ethanol - blue non-diffusible pigment P. fluorescens IV d = positive for more than 10 % but less than 90 % of 37 varied among samples (Table 6). No two samples were identiCal in both. At least five genera were: represented in the total "number of denitrifiers, as shown in Table 7. Pseudomonas spp. dominated by comprising 65.3 % of the total isolated. There appeared to be 37 distinct species or strains among the 147 isolates characterized. A complete list of these can be found in Appendix. Table 8 lists the major species represented along with their occurrence in the 19 samples. Although Pseudomonas was the predominant genera,- Alcal- igenes faecalis was the predominant species, and occurred in the most samples. Pseudomonas fluorescens II also occurred in high numbers. Identified species found in fewer numbers than those listed in Table 8 included Pseudomonas aureofaciens, and Pseudomonas solanacearum. Temperature Relationship From Table 2, soil and sediment samples obtained directly from the environment Can be divided into two main groups based on mean annual temperature. One group is described as temperate, representing samples with a mean annual temperature of 20 C and below, and the other trop- ical, representing samples with a mean annual temperature of 20 C and above (Table 9). By observing the ability of denitrifying isolates to grow at 4 C and 41 C, a relation- ship was determined between the mean annual temperature of the sampling location and the temperature at which the 38 Table 6. Denitrifiers Isolated from Samples. Number that Den- Sample Organisms Isolated itrified After Purification Michigan(P4) Alcaligenes faecalis 1 November Pseudomonas fluorescens II 2 Pseudomonas f1uorescens(?) 1 Pseudomonas sp. type 1 l Pseudomonas sp. type 2 _2 7 Poultry manure Alcaligenes faecalis 14 Connecticut 1 Alcaligenes faecalis 3 unknown type 3 _2 5 California Alcaligenes faecalis 1 Pseudomonas fluorescens II 5 Corynebacterium sp. 1 Flavobacterium sp. _1 8 Minnesota Alcaligenes faecalis l Pseudomonas aureofaciens 3 Pseudomonas fluorescens II 16 Pseudomonas sp. type 2 5 Pseudomonas sp. type 4 l Pseudomonas sp. type 5 4 Pseudomonas sp. type 6 2 Pseudomonas sp. type 7 _l 33 Brazil Pseudomonas sp. type 18 4 Kansas Pseudomonas fluorescens 1 Argentina(P) Pseudomonas fluorescens IV 3 . Flavobacterium sp. 5 Pseudomonas sp. type 19 _l 9 39 Table 6. (continued) Number that Den- itrified After Purification Sample Organisms Isolated Argentina(SP) Michigan(Pl) May Philippine Michigan(muck) Michigan(P4) May Nigeria(C) Alcaligenes faecalis Pseudomonas fluorescens II Pseudomonas f1uorescens(?) Pseudomonas stutzeri Bacillus sp. Pseudomonas Pseudomonas sp. type 11 sp. type 23 unknown type 21 unknown type 22 unknown type 24 Alcaligenes faecalis Pseudomonas fluorescens II Pseudomonas fluorescens IV Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas sp. type 8 sp. type 9 sp. type 10 sp. type 11 aeruginosa Pseudomonas Pseudomonas sp. type 11 stutzeri Pseudomonas Pseudomonas Pseudomonas f1uorescens(?) sp. type 12 sp. type 13 unknown type 3 Alcaligenes faecalis Alcaligenes eutrophus ngynebacterium sp. Pseudomonas Pseudomonas Pseudomonas sp. type 11 sp. type 19 sp. type 20 [—4 N M‘HF‘FHHBJPHQFJH |'-" m hawtahndnam .8 else \nhanlwrdhud b hthdh' 40 Table 6. (continued) Number that Den- Sample Organisms Isolated itrified After Purification Nigeria(R) Alcaligenes faecalis 1 Pseudomonas solanacearum l Pseudomonas sp. type 11 1 Pseudomonas sp. type 16 l Pseudomonas sp. type 17 _l 5 Taiwan Alcaligenes faecalis 1 Pseudomonas sp. type 16 2 Pseudomonas sp. type 14 _l 4 Michigan(WG) Pseudomonas stutzeri 2 Alcaligenes faecalis _1 3 Texas Pseudomonas sp. type 11 1 Pseudomonas sp. type 25 _2 3 Louisiana Pseudomonas sp. 14 1 Pseudomonas sp. 16 1 unknown type 15 _l 3 Table 7. Major Genera Recovered. Genera Percent of Total Pseudomonas 65.3 Alcaligenes 23.8 Flavobacterium 4.1 Bacillus 1.4 Cogynebacterium 1.4 unknown others 4.1 41 Table 8. Major Species Recovered. Species Pefiii‘i °f 0538:3352.“ Alcaligenes faecalis 23.1 11 Pseudomonas fluorescens II 17.7 5 Pseudomonas sp. type 2 4.8 2 Pseudomonas sp. type 11 4.1 6 Flavobacterium sp. 4.1 2 ' Pseudomonas aeruginosa 4.1 1 Pseudomonas fluorescens IV 3.4 3 Pseudomonas stutzeri 3.4 3 Pseudomonas f1uorescens(?) 2.7 3 Pseudomonas sp. type 16 2.7 3 Pseudomonas sp. type 5 2.7 1 Pseudomonas sp. type 18 2.7 1 Table 9. Samples from Tropical and Temperate Locations.1 Soils From Locations With Mean Annual Tem- peratures of 20 C Soils From Locations With Mean Annual Tem- peratures of 20 C and And Above Below Nigeria(R) Argentina(SP) Nigeria(C) Argentina(P) Philippines Minnesota Louisiana California Brazil Michigan(muck) Taiwan Michigan(WG) Texas Michigan(Pl) May Michigan(P4) May Michigan(P4) November Kansas 1Samples were excluded which were incubated under un- natural conditions or of which denitrifiers were not isola- ted. 42 isolates from the samples can grow. Of the 95 denitri- fiers isolated from the temperate samples, 68.4 % grew at 4 C, 9.5 % grew at 41 C, and 22.1 % grew only at 28 C. Of the 33 denitrifiers isolated from the tropical soils, none grew at 4 C, 66.7 % grew at 41 C, and 33.3 % grew only at 28 C. No denitrifiers were isolated that could grow at both 4 C and 41 C. A species relationship can also be observed with Pseudomonas fluorescens II and Alcaligenes faecalis, which grow at 4 C and 28 C, but not at 41 C. Of the total number of both species isolated from the two temperature groups, all of the Pseudomonas fluorescens II isolates and 85.0 % of the Alcaligenes faecalis isolates were from the temper- ate samples. DISCUSSION Development Of A Medium And.Method For The Isolation And Enumeration Of Denitrifyinngacteria Before actual isolations were attempted, it was desirable to know that the medium chosen for use would give the highest possible counts and yet be as selective as possible for denitrifying bacteria. The importance of soil extract for the enumeration of soil bacteria has been emphasized by many soil microbiologists (29). A broad range of heat-stable soil nutrients is provided in small amounts by soil extract. Yet the amount of carbon is low enough to prevent perceptible antibiotic and organic acid production and the growth of spreaders. However, the use of soil extract has been criticized by many. Kfister found the greatest numbers appeared on soil extract pre- pared from the soil that was being examined (22). Thus, a soil extract medium prepared from one soil may not be suitable for the growth of bacteria from another soil. Bacteriocidal substances have also been reported in ex- tracts of soil (22). In this study the soil extract- yeast extract media did not yield as high a population of denitrifiers as did nitrate broth and thus it was not used. On agar, diffusibility of metabolites away from col- onial growth is limited. The metabolism of an organism 43 44 may produce a pH change that is toxic and lowers the organ- isms viability. The process of denitrification generates OH- ions. In unbuffered broth cultures of denitrifying bacteria, I observed pH increases from 7.2 to 8.9 for cer- tain isolates, which resulted in their death. The need for a buffer to neutralize such an effect is evident. However, my testing on the use of phosphate buffer demon- strated that it had an inhibitory effect on denitrifica- tion. When autoclaved, the buffer may have precipitated trace divalent cations, needed by the nitrate reductase (32). I The medium and method of inoculation excluded growth of many unwanted microorganisms. Since inoculations were carried out under aerobic conditions, the death of some non-sporeforming obligate anaerobes would occur. Incuba- tions under anaerobic conditions would not permit the growth of aerobes incapable of anaerobic growth with nitrate. Since the medium was carbohydrate free, carbo- hydrate fermenters could not grow. Because no reducing agent was added to the medium, the redox would not be low enough to permit the growth of many spore-forming obligate anaerobes. Therefore, the medium was considered selective for organisms capable of respiring with nitrate. However, 37.9 % of the isolates tested were unable to utilize the nitrate in the broth after isolation on agar. Many of these produced only pinpoint colonies on agar and were thought possibly to be microaerophiles capable of growing on trace 45 amounts of O2 trapped in the agar. Although the medium and method for denitrifier iso- lations seemed satisfactory, there are problems. The incu- bation period was limited to a five day maximum because of the over-growth of certain organisms. Yet, the poss- ibility does exist that slow-growing organisms would not be visible in five days. Such denitrifiers would be missed. Because of their slow growth, their contribution to denitrification would likely be minimal. Many organ- isms that grew on agar would not grow in broth. Such organ- isms could have been microaerophilic, as discussed earlier. It is also possible that such organisms which were able to grow well on agar but not in broth, were inhibited by volatile fatty amines released from the polyurethane foam stoppers during autoclaving (5) or needed H2 (present in glove box atmosphere), which is insoluble in broth. An unresolved troublesome problem was the loss of the denitrifying ability by certain isolates after purification. Most of the organisms were still able to grow anaerobically with nitrate, reducing it to nitrite, but were unable to form gas. This suggests a lack of nitrite reductase. Another possibility is an alternate dissimilatory pathway after nitrite formation. Fewson and Nicholas (17) state (that hydroxylamine and ammonia can be formed during nitrate dissimilation. This could be an explanation for the obser- vation that certain organisms depleted the broth of nitrate, yet produced neither nitrite, ammonia, or gas. Isotopic 46 studies with 15N nitrate could prove the existence of such an alternate pathway. An unsuitable medium or an unstable genetic character could also explain the loss of denitri- fying ability. Commonality Of Denitrifier Isolates Four of the 14 known genera of denitrifying bacteria (32) were identified among the 147 types isolated and charac- terized from 25 samples examined. Since Propionibacterium spp. are only found in dairy products or the skin and in- testinal tract of man and animals, Moraxella spp. are parasitic on the mucous membranes of warm-blooded animals and man, Halobacterium spp. require very high salt concen- trations for survival, and Achromobacter spp. are now in other genera, these genera were not expected to be isolated. Also, since culturing conditions eliminated the possibil- ity of autotrophs, Thiobacillus spp. were not expected. Therefore, the following genera were possibilities for the identification of denitrifiers isolated: Alcaligenes, Ba- cillus,Chromobacterium, Corynebacterium, Hyphomicrobium, Micrococcus, Pseudomonas, Spirillum, and Xanthomonas. However, only Alcaligenes, Bacillus, Corynebacterium, and Pseudomonas were identified of those known genera of den- itrifiers. A Flavobacterium sp. was.also identified as a denitrifier, which has not previously been reported. Flavobacterium spp., however, have been reported to reduce nitrate to nitrite (32). The high proportion of 47 the isolates being Pseudomonas spp. was not surprising because of their ability to utilize such a great number of carbon sources and their well-known prevalence in soils. The prevalence of Alcaligenes was surprising since members of this genus had not been thought to be common soil den- itrifiers. Although they are defined as separate genera, Alcaligenes and Pseudomonas are very similar. Almost all members of both genera are gram negative rods, obligate respirers, oxidase and catalase positive, able to use acetate as a sole carbon source, motile, and have a G + C content of DNA from 58 to 70 moles % (10). They are dif- ferentiated on the number and location of flagella - for Pseudomonas, single polar, and for Alcaligenes, peritri- chous. However, Bergey's Manual (10) describes Alcaligenes as possessing one to four (occasionally up to eight) peritrichous or degenerate flagella. The possibility of an Alcaligenes sp. having a: single subpolar flagella would make it virtually impossible to distinguish from a pseudomonad. Almost two-thirds of the denitrifier isolates were identified to the species level. Nine separate species were identified. Most of the 16 described strains of denitrifiers recognized in Bergey's..Manual (10) in the genera of Pseudomonas and Alcaligenes were represented. However, Pseudomonas fluorescens biotype II and Alcaligenes faecalis were numerically dominant and were most frequently observed from the samples. Pseudomonas flugrescens II is 48 found in soil and water and generally considered to be saprophytic (10). Some strains of Pseudomonas fluorescens are known to be common inhabitants of the rhizosphere (35). The presence of denitrifying strains in anaerobic ndcro- sites of the rhizosphere may greatly affect plant product- ivity by causing a localized depletion of nitrogen. The Alcaligenes faecalis strains isolated were very similar to the strain described by Pichinoty et a1. (33). The major differences observed were the inability of my strains to utilize D-saccharate, ethanol, and citrate as sole carbon sources. The high occurrence of Alcaligenes faecalis leads one to question its source. It is a species of a genus widely distributed in decomposing organic matter and also found in the intestinal tract of vertebrates. Its origin could have been fecal because of its wide dis- tribution in these agricultural and natural soils. However, it is more likely an indigenous soil inhabitant. Many of the most commonly studied denitrifying bac- teria were not isolated or isolated in very small numbers. Paracoccus (formerly Micrococcus) denitrificans and Pseu- domonas denitrificans (ATCC 19244) and Pseudomonas denit- trificans (ATCC 13867) were not identified from any of the 25 samples. Such an observation leads one to believe that they play a minor role in the environment. Douderoff et. a1. (14) examined many previously isolated strains of pseudomonads to determine the validity of P. denitrificans as a species and found no other 49 organisms related to either of these two dissimilar species. Due to the absence of other related strains, he proposed that the name Pseudomonas denitrificans be abandoned. Since no new isolates from diverse samples were found in this study which were related to either of the P. denitri- ficans, the proposal of Douderoff (14) is supported. Correlations Between Samples, And MPN's And Isolates There appears to be very little correlation. between sample environmental parameters, population densities, and identity of organisms isolated. Only the environmental extremes--desert, acid rain forest--had any effect on population densities. Almost all of the MPN's for denit- 5 6 organisms-g.l when pH trifiers ranged from 10 to 10 values were between 4.42 and 7.84. Only at 3.84 and 8.21 were MPN's less than 104 organisms-9-1. Certain isolates did appear only in particular pH ranges, e.g. Pseddomonas fluorescens IIwas mainly isolated in samples with a pH range of 6.94 to 7.34. -A1ca1igenes faecalis was isolated from samples representing a broad pH range. The most dramatic correlation observed was between the mean annual temperatures of the sample locations and the ability of isolates to grow at certain temperatures. Either the temperatures of the environment select for certain organisms or the organisms themselves adapt to a particular temperature range. The fact that none of the isolates from the tropical samples were able to grow at 50 4 C is reasonable, since there is no advantage in adapting to a temperature the organism will never encounter. Whereé as, there is a competitive advantage for temperate isolates to be able to grow at 4 C, since they are exposed to 4 C but not 41 C. Improvements It is probable that the media and isolation procedure used in this study could be further improved. For higher counts and possibly more representative isolates, a soil extract medium made from the soil in which enumerations and isolations are to be done could be employed. Also, adjusting the pH of the medium to the same pH as the soil would best mimic the actual environment, therefore allowing the growth of those denitrifiers unable to grow at neutral pH. However, the incubation period would have to be much longer, since growth would be much slower under such Cir- cumstances. The use of nitrite, instead of nitrate, in the initial isolation medium, would eliminate the organ- isms able to reduce nitrate to nitrite but no further. This approach would only work if a concentration of nitrite could be found that would not be toxic to the soil denitri- fiers. One might also be able to use N20 as the electron acceptor and thus only recover denitrifiers. LITERATURE CITED 10. 11. LITERATURE CITED Aaronson, S. 1970. Experimental Microbial Ecology. Academic Press. New York. Adel, A. 1946. A possible source of atmospheric N20. Science 103: 280. Adel, A. 1951. Vertical distribution and origin of atmospheric nitrous oxide. Astron. J. 56: 33-34. Aranki, A., S.A. Syed, S.B. Kenney, and R. Freter. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 11: 568-576. Bach, J.A., R.J. Wnuk, and D.G. Martin. 1975. Inhi- bition of microbial growth by fatty amine catalysts from polyurethane foam test tube plugs. Appl. Micro- biol. 29: 615-620. Black, C.A. 1965. Methods of Soil Analysis, Part 1, Physical and Mineralogical Properties, Including Statistics of Measurement and Sampling. American Society of Agronomy, Inc. Madison, Wis. Black, C.A. 1965. Methods of Soil Analysis, Part 2, Chemical and Microbiological Properties. American Society of Agronomy, Inc. Madison, Wis. Breed, R.S., E.G.D. Murray, and N.R. Smith. 1957. Bergey's Manual of Determinative Bacteriology. 7th Ed. Williams and Wilkins Co., Baltimore. Broadbent, F.E., and F. Clark. 1965. Denitrification, p. 347-379. £2 Soil Nitrogen. Bartholomew, W.V. and F.E. Clark. American Society of Agronomy, Madison, Wis. Buchanon, R.E., and N.E. Gibbons. 1974. Bergey's Manual of Determinative Bacteriology. 8th Ed. Williams and Wilkins Co., Baltimore. Burns, R.C., and R.W. Hardy. 1975. Nitrogen Fixation in Bacteria and Higher Plants. Springer-Verlag, New York, Heidelberg, and Berlin. 51 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 52 Caldwell, D.E., and J.M. Tiedje. 1975. A morpholog- ical study of anaerobic bacteria from the hypolimnia of two Michigan lakes. Can. J. Microbiol. 22: 362- 376. Colwell, R.E., and W. J. Wiebe. 1970. "Core" charac- teristics for use in classifying aerobic, hetero- trophic bacteria by numerical taxonomy. Bull. Ga. Acad. Sci. 22: 165-185. Doudoroff, M., R. Contopoulou,R. Kunisawa, and N.J. Palleroni, 1974. Taxonomic validity of Pseudomonas denitrificans (Christensen). Init. J. Syst. Bacter- 101. 22: 294-300. FAO UNESCO. 1971. Soil Map of the World (converted to the 7th Approximation). Paris. Ferguson, M., and E.B. Fred. 1908. Denitrification: The effect of fresh and well-rotted manure on plant growth. Virginia Agr. Exp. Sta. Ann. Rep. 1908: 134-150. Fewson, C.A., and D.J.D. Nicholas. 1961. Utilization of nitrate by micro-organisms. Nature 190: 2-7. Focht, D.D., and H. Joseph. 1973. An improved method for the enumeration of denitrifying bacteria. Soil Sci. Soc. Amer. Proc. 22: 698-699. Gayon, E., and G. Dupetit. 1886. Resherches sur la reduction des nitrates par 1es infiniments petits. Soc. Sci. Phys. Nat. Bordeaux, Sér. 3, 2, 201-307. Geiger, R. Mean Annual Precipitation Map. Justus Perthes. Darmstadt, Germany. Gordon, R.E., and J.M. Mihm. 1959. A comparison of four species of mycobacteria. J. gen. Microbiol. 22: 736-748. Gray, T.R.G., and D. Parkinson. 1968. The Ecology of Soil Bacteria. University of Toronto Press. Toronto. Gray, T.R.G., and S.T. Williams. 1971. Soil Micro- organisms. Hafner Publishing Co., Inc. New York. Haak, H. Physikalischer Wandatlas I. Abteilung: Klima und Wetter l. Linien gleighen Warme im Jahr. Gotha: Justus Perthes. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 53 Holdeman, L.V., and W.E.C. Moore. 1973. Anaerobe Laboratory Manual 2nd Ed. Virginia Polytechnic In- stitute and State University Anaerobe Laboratory, Blacksburg, Va. Johnson, L.F., and E.A. Curl. 1972. Methods for Re- search of Soil-Borne Plant Pathogens. Burges Pub- lishing Co., Minneapolis, Minn. King, E.O., M.K. Ward, and D.E. Raney. 1954. Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab & Clin. Med. 22: 301-307. Kluyver, A.J. and H.J.L. Donker. Die Einheit in der Biochemie. Chem. Zelle u. Gewebe 13, 134-190. Lochhead, A.G., and M.O. Burton. 1956. Importance of soil extract for the enumeration and study of soil bacteria. 6th Int. Congr. Soil Sci. C, 157. Manual of Microbiological Methods. 1957. Society of American Bacteriologists. MCGraw-Hill Book Co., Inc. New York. Nelson, W.W. and J.M. MaCGregor. 1973. Twelve Years of Continuous Corn Fertilization With Ammonium Ni- trate or Urea Nitrogen. Soil Sci. Soc. Amer. Proc. 37: 583-586. Payne, W.J. 1973. Reduction of nitrogenous oxides by microorganisms. Bact. Rev. 21: 409-452. Pichinoty, F., M. Mandel, B. Greenway, et J. Garcia. 1975. Isolement a partir du sol et étude d'une bactérie dénitrifiante appartenant au genre Alcal- igenes. C.R. Acad. Sc. Paris, t. 281. Prakasam, T.B.S. 1972. Microbial nitrification and denitrification in concentrated wastes. Wat. Res. 9: 859-869. Sands, D.C., and A.D. Rovira. 1971. Pseudomonas fluorescens Biotype G, the dominant fluorescent pseudomonad in South Australian soils and wheat rhizospheres. J. appl. Bact. 34(1): 261-275. Schmidt, E.L. 1974. Quantitative autecological study of microorganisms in soil by immunofluorescence. Soil Sci. 11: 141-149. Standard Methods for the Examination of Water and Waste- water. 1971. Amer. Public Health Assoc. 13th Ed. Washington, D.G. 38. 39. 40. 41. 42. 43. 54 Stanier, R.Y., N.J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: a taxonomic study. J. gen. Microbiol. 43: 159-271. Starr, J.L., and J.Y. Parlange. 1975. Nonlinear denitrification kinetics with continuous flow in soil columns. Soil Sci. Soc. Amer. Proc. 39: 875- 880. Tiedje, J.M., and B.B. Mason. 1974. Biodegradation of nitriloacetate (NTA) in soils. Soil Sci. Soc. Amer. Proc. 38: 278-283. Thornley, M.J. 1960. The differentiation of Pseudo- monas from other gram negative bacteria on the basis of arginine metabolism. J. Appl. Bact. 23: 37-52. Turner, A.W. 1954. Bacterial oxidation of arsenite I. Description of bacteria isolated from arsenical cattle-dipping fluids. Aust. J. Biol. Sc. 7: 452- 478. Wallingford, G.W., L.S. Murphy, W.L. Powers, and H.L. Manges. 1975. Effects of Beef-Feedlot Manure and Lagoon Water on Iron, Zinc, Manganese, and Copper Content in Corn and DTPA Soil Extracts. Soil Sci. Soc. Amer. Proc. 39: 482-487. APPENDIX Table 10. Percentages of Sand, Silt, and Clay in the Soil Samples as Determined.by the Hygrometer Method.* Soil % Sand % Silt % Clay Minnesota 30 55 15 California I 35_ 47 18 Argentina(SP) 42 39 19 Argentina(B) 33 54.5 12.5 Texas 43 27 30 Argentina(P) 38 '41 21 Brazil 40 29.5 30.5 Nigeria(C) 15 76 9 Nigeria(R) 10 85 5 Columbia 36 42 22 Philippines 27 58 15 Taiwan 32 57 11 Louisiana ‘ 41 44 15 .Utah 37 4 50.5 12.5 Kansas 28 ' 64 8 * The hygrometer method depends on complete dispersion of soil particles. Due to the extreme diversity of the soils studied, this could not be achieved by a standard method for all cases. Therefore, some of the above percentages are probably incorrect. 55 56 Explanation of Table 11 Table 11 contains the data of the 52 properties ex- amined for the identification of the denitrifying isolates. Positive and negative designations were + and 0 respective- ly; 3 means the test was not done. Test were coded by number as follows: gram reaction motility oxidase catalase acetate . gelatin hydrolysis starch hydrolysis casein hydrolysis fluorescein (medium B) 10 blue phenazine (peptone-glucose) 11 D-glucose 12 sucrose 13 D(+)trehalose 14 L-arabinose 15 D-fructose 16 D-arabinose 17 ethanol 18 growth at 41 C 19 propionate 20 propylene glycol 21 L-asparagine 22 D-sorbitol 23 geraniol 24 D-xylose 25 DL-arginine 26 D-ribose 27 maltose 28 D(+)cellobiose 29 meso-inositol 30 sarcosine 31 B-alahine 32 p-hydroxybenzoate (base) 33 p-hydroxybenzoate (acid) 34 2-keto-gluconate 35 saccharate 36 growth at 4 C 37 arginine dihydrolase 38 arsenite oxidation 39 PHB granules 40 citrate \DmxlmmbWNH 57 Pigment color abbreviations were: unpig - unpigmented, pale yel - pale yellow, YG - yellow-green, BG - blue-green, and d - diffusible. The pigments without the d designation were not diffusible. Cell dimension were in pm. For col- onial characteristics, column 1 was size in mm, with p for pinpoint; 2, form, where c - circular and i - irregular; 3, elevation, where c - convex, u - umbonate, p - pulvinate, r - raised, and f - flat; 4, margin, where u - undulate, en - entire, * - not describable, and er - erose; 5, surface, where s - smooth, ru - ruffled, and r0 - rough; 6, texture, where b - buttery, d - dry, and m - mucoid; 7, light refraction, where g - glistening, t - translucent, d - dense, and o - opaque. The number 8 designates cell groupings, with s - singular and c - chains. 58 Table 11. Results of 52 Properties Tested. Tests Sam 1e Isolate 1111111111222222222233333333334 P Number 1234567890123456789012345678901234567890 Lake 4 November 4 0++++000000000000000+000000000+0000+00+0 6 0++++00000+00++0+0+0++0+++00+++00+++00++ 12 0++++00000+00++0+0+0++0+++00+++00+++000+ l3 o+++++o++o+++++++o++++++++o++o++o++++o0+ 14 0+++++0+00+00++0+0+0+00+0+0000+0+0++00+0 15 0+++++0++0+00+++00+0+00+++0+++++0++++00+ 16 0+++++0++0+++++++0++++++++0++0++0++++00+ Nitrified 17 0++++000000000000000000000000000000+0000 poultry 18 0++++00000000000000+000000000000000+0000 manure 19 0++++000000000000000000000000000000+0000 20 0++++00000000000000++00000000000000+0000 21 O++++00000000000000++00000000000000+0000 22 0++++000000000000000000000000000000+0000 24 0++++000000000000000000000000000000+0000 25 0++++000000000000000000000000000000+0000 26 0++++000000000000000000000000000000+0000 27 0++++000000000000000000000000000000+0000 28 0++++0000000000000+O00000000000000000000 29 0++++000000000000000000000000000000+0000 30 0++++00000000000000+000000000000000+0000 31 0++++000000000000000+0000000000000000000 Connecticut 1 36 v+++0+00000000000+00000000000000000000+0 37 0+++000000000000000000000000000000000000 39 v++00000000000000000000000000000000000+0 40 0+++000000+00000000000000+00000000000000 41 0++++00000000+0+000000000000000000000000 California 42 o+++++o++++++++++o++++++++o+++++o++++o0+ 43 0++++0000000000000+0+0000000000000++000+ 44 0+++++0++++++++++0++++++++0+++++0++++00+ 45 0+++++00+++++++++0++++++++0+++++0++++00+ 46 00+++++000+++++0000000++00++0000000+0000 47 o+++++o++o+++++++o++++o+++oo+o++o++++oo+ 48 000++00000+00+0+0000+00000000000000000+0 49 o+++++o++o+++++++o++++o+++o+++++o++++o0+ Minnesota 51 o++++oooo0+0+++o+o++++o+++oo+++oo+++oo++ 52 o+++++o++o+++++++o++++++++oo+o++o++++00+ 53 0+++++0++0+++++0+0++++++++0+++++0++++00+ 54 0++++00000+00++o+0++++++++00+++0o+++000+ L ‘ ~ - _4L__ 59 Table 11. (continued) Tests Sample Isolate 1111111111222222222233333333334 Number 1234567890123456789012345678901234567890 Minnesota (continued) 55 0++++000+0+o+++++0+++00+++00+0+00++++00+ 56 o++++o00oo+o+++o+o+o++o+++oo+++oo+++oo++ 57 0++++000+0+0+++++0+++00+++00+0+00++++00+ 58 0++++000+0+0+++++0+++0++++000++00++++00+ 59 0+++++0++0+++++0+0++++++++00+0++0++++00+ 60 0+++++0++0+++++0+0++++++++00+0++0++++00+ 61 0+++++00+0+++++000++++++++00+0++0++++00+ 62 0+++++0++0+++++++0++++++++00+0++0++++00+ 63 0+++++0++0+++++0+0++++++++00+0++0++++00+ 64 o+++++o++o+++++++o++++0+++oo+o++o++++oo+ 65 0++++000000000000000+00000000000000000++ 66 0+++++0++0+++++0+0++++0+++00+0++0++++00+ 67 0+++++0++0+++++++0++++0+++0+++++0++++0++ 68 o+++++o++o+++++o+o++++++++o+++++o++++o++ 69 0+++++0+00+++++0+0+++0++++00+0++0+++00++ 7o 0+++++o++0+++++++0++++++++00+o++o++++00+ 71 0+++++o+oo+++++o+o++++++++oo+o++o+++oo0+ 72 0+++++0++0+++++++0++++0+++00+0++0++++00+ 73 0+++++0++0+++++++0++++++++0+++++O++++00+ 74 o+++++o+oo+++++o+o++++++++oo+o++o++++o++ 75 o+++++oo+o+++++o+o++++++++o+++++o++++o0+ 78 o++++o000o+o+++o+o+++o++++o++o+oo++++oo+ 79 0+++++0++0+++++++0++++++++0++0++0++++00+ 80 0++++000+0+00++++0+++0++++0+++++0+++000+ 81 0++++00000++o++0+0++++++++0++++00+++00++ 82 0+++++0+0++++++0+0++++++++00+0++00+++0++ 83 0++++00000+0+++0+0+++++++++0+++00+++00++ 84 0+++++0++0+++++++0++++++++++++++0++++00+ 85 0+++++o+00+o+++++o+++o++++++++++0+++00++ Lake 1 May 86 0++0+000000000000000000000000000000000+0 87 o++++ooooo+oo+oo+oo++++o+ooooo+oooo++ooo 89 0+++++00+0++0++++0++++++++00+0++0++++00+ 90 0++0+000000000000000000000000000000000+0 91 0+++000000000000000000000000000000000030 97 0+0+0+0+000+00000+00+0000000000000000000 98 o++++ooo+o+++++++o++++++o++oooooo+ooo00+ 99 0++++000000000000000000000000000000000+0 101 0++++000000000000+00+0+00000000+000000+0 102 O++++000000000000000+00000000000000000+0 60 Table 11. (continued) Sample Isolate Number Tests 1111111111222222222233333333334 1234567890123456789012345678901234567890 Lake 1 (continued) 103 104 105 106 107 881 Lake 4 May 108 110 111 882 Louisiana 114 115 1181 Brazil 126 129 133 135 Nigeria(R) 137 141 143 144 145 Nigeria(C) 1471 148 149 151 153 154 155 Philippines 156 162 163 0++0+00000000+00+00++0+0+00++0+0000+0000 0+++0000000000000000000000000000000000+0 0+++++0++++++++++0++++++++0++0++0+++00++ 0++++00000000000000+0000000000000+0000+0 o++++oooo0+++++++o++++o+++++++++oooooo++ 03++0000000000000000000000000000000000+0 v+0+0++0000000000+00+00000000000000000+0 o+++++o+oo+++++o00+o+oo+++oo+o++o+++oo++ o+++++o++o+o+++ooo++++o+++oo+o++o+o++oo+ 0+++++0000++0++000+0++0++++++0000+0+00++ 0++0+00000+++0+00++0+000+0+000000++000+0 +00+0000000000000+0000000000000000000000 0+++++0000+0+++00++0+00+000000++0++000++ 0+++++0000+0+++00+00++0+000+0+++0++000++ 0+++++0000+0+++00+00+00+000+0+++0++0000+ o+++++oooo+o+++oo+00++o+oo0+00++o++000++ 0++0++0000+0+++00++0++0+000+00++0++000++ ooo++ooooooooo+o+o+++o++++ooo+++o++ooo++ 0+00+00000+0+++00++0+0++000+0+++0++0000+ 0++++00000++++++00++++0++++++0+0+00000+0 0+++0000000000000000000000000000000000++ 03+3000000++00+00+0030+00000000000003333 00000++000++0000000000+++++++0000++000+0 0+0++00000000000+0+0+00000000+0+00+000++ 0++++00000++++++0000++0++++++0++000000+0 o++o++oooo+ooooooo+ooooooo++oooooooooooo 0++0++000000000000+0000000++000000000000 0++++00000+00++000+++0+0+00000++0000+00+ 0++++00000+000+0+++++000000000+0+00000+0 0+++++0++0+0+++++++++00+++000+++0+00+00+ 0+++++0++0+0+++++++++00+++000+++0+00+00+ o++++ooooo+++++++o++++o+++++++++oooooo++ 61 Table 11. (continued) Sample Isolate Number Tests 1111111111222222222233333333334 1234567890123456789012345678901234567890 Philippines (continued) . 164 165 166 167 Taiwan 171 172 173 174 Argentina(P) 175 176 177 178 179 180 183 184 185 Argentina(SP) 188 189 190 191 192 193 195 196 199 202 204 205 Kansas 206 Michigan(WG) 220 221 223 0+0+++0++0+0+++++++++00+++000+++0+00+00+ 0+++++0++0+0+++++++++00+++000+++0+00+00+ o+++++0++0+0+++++++++00+++000+++0+00+00+ 0+++++0++0+0+++++++++00+++000+++0+00000+ 00000++0000000000+00000000000000000000+0 0++++00000+o+++00++0+0+++0000+++o++000++ 0++0000000+++0+00000+000+033333333333333 0++0++0000+0+++00++0+00+00000+++0++000++ 00++0++000++0++0000000++00++0000000+0000 00++0++000++0++0000000++00++0000000+0000 00++0++000++0++00000000+00++0000000+0000 00++0++000++0++00000000+00++0000000+0000 0++++00000000+000000000000++000+00+00000 00++0++000++0++00000000+00++0000000+0000 0+0+++0++++++++000+0++++++00+0++o++++0++ o+o+++o++++++++ooo+o++++++oo+0++o++++o++ 0+o+++o++++++++++o+o+++++++++o++o++++o++ 0++++00000+++++++0++++0++++++0++000000+0 00000++0000000+000000+00000000000000+0+0 0++++000+0+00++++0++++++++0000+00++++0++ 0+++000000000000000000000000000000+000++ v000000000+++++000000+0+0++0+000000000+0 v0000+00000000+000000+0+0+00+000000000+0 0++++0+000+00++0++00000++0+00+0+0000000+ 0++++000+0+00++++0+++0++++0000+00++++0++ v00+0++000++0++00000+++00++0+00000+000+0 00000++000++0++00000++00++000000000000+0 0+0000+000+++++00+00000+00+0000000000000 0+++++0++0+++++0+0++++++++00+0++0++++00+ 0+++++0+++++0++++o++++++++++++++oo+++o++ 0++++o+ooo+oo++o++o++oo+o++oo+o+oo00000+ 0++++0+000+00++0++0++00+0++00+0+0000000+ 0+++++000000000000+0000000000000000000+0 62 Table 11. (continued) Sample Isolate Number Tests 1111111111222222222233333333334 1234567890123456789012345678901234567890 Michigan (muck) 224- 2312 Texas 232 233 234 Stock Cultures 991 992 993 994 995 996 997 998 999 o++++o+ooo+oo++o++oo+o++o++00+o+oooo000+ 0++++0+000+00++0++0++00+0++00+0+0000000+ 0+o++ooooooooo+00++o+oooooooo++ooo+ooo++ o+o++ooooooooo+oo++o+oooooooo++ooo+ooo++ o++++00000+++++++000++0++++++0+0+00000+0 0++++00000+000++00+0+0+0+00000++0000+00+ 0+0+++0++0+++++000+0+00+++00+0++0++++00+ 0++0+0+000+00++0+++++00++0000++00000000+ 0++++00000000000+000+00000000+000000000+ 0+++++00+0+++++++0++++++++00+0++0++++00+ o+++++oo+o+oo+++++++++o+++ooo+++o+oooo++ 00+++00000+++++0+0++++00+++0+0++0+000000 0++++0+000+000+00+0000++00+00+000000000+ 0++++0+000+00000+00++00+00+00+000000000+ 63 Table 11. (continued) Colonial Isolate Cell Pigment Cell Characteristics Number Morphology Color Dimensions 1 2 3 4 5 6 7 8 4 rod unpig .8 x. 2.5 1 c c en 3 b g s 6 rod white .8 x 4.0 2 c c en 3 b g s 12 rod cream .8-1.2x2.5-3.3 2 c c en 8 b g s 13 rod cream .8 x 1.7-3.3' 1 c c en 5 b g s 14 rod cream .8 x 3.3-4.1 4 c c en 3 b g s 15 rod cream .8 x 2.5 5 i c en 8 b g s 16 rod cream .8 x 1.7-3.3 4 i c en 3 b g s 17 rod unpig .8 x 2.0-2.9 3 c u er ru b t s 18 rod unpig .8 x 2.5 l c c en 3 b g s 19 rod unpig .8 x 2.0-2.5 2 c u er ru b t s 20 rod unpig .8 x 1.7-2.9 1 c c er s b g s 21 rod unpig .8 x 2.0-2.9 1 c c en 3 b g s 22 rod unpig .8 x 1.7-3.3) 3 c u er ru b t s 24 rod unpig .8 x 1.7-2.0 3 c u er ru b t s 25 rod unpig .8 x 3.3 3 c u er ru b t s 26 rod unpig <8-.8x1.7-2.5 3 c u er ru b t s 27 rod unpig .8 x 2.5 3 c u er ru b t s 28 rod unpig .8 x 2.9—3.3 ].c c en 3 b g s 29 rod unpig .8 x 1.7-2.5 2 c c en 3 b g s 30 rod unpig 1.2 x 2.9 l c c er s b g s 31 rod unpig .8-1.2x2.5-3.0 3 c c en 5 b g s 36 rod cream .8-1.2x2.5-3.0 2 c c en 5 b g s 37 rod unpig 1.2x2.5-2.9 2 c c er ro b o s 39 rod unpig .8x2.9-4.1 1 c c en ro b g s 40 rod unpig <8-.8x2. 5- 2. 9 1 c c en 3 b g s 41 rod unpig .8 x 2. 0 -p c c en 5 b g s 42 rod cream .8 x 2.0- 3.7 :2 c c en 5 b g s 43 rod unpig .8 x 1.7- 2.5 «3 c c en 3 b g s 44 rod pale yel .8 x 2.0-3.3 2 c c en 3 b g s 45 rod pale yel<8-.8x2.5-3.3 l c c en 3 b g s 46 rod ye1.4 x 3.7- 4.1 1 c c en 3 b g s 47 rod pale yelci. 8 x 1.7- 4.1 4 c c en 8 b g s 48 pleomorph cream “ p c c en 3 b g - 49 rod white .8- 1. 2x2. 9- 4. 1 3 c c en 3 b g s 51 rod gray 1.7 x 3.7 2 c c en 5 b g s 52 rod tan .8-1:2x2.0-4.1. 4 i c en 3 b g s 53 rod pale yel .8x2.5-3.3 2 c c en 5 b g s 54 rod cream 1.2x2.0-3.3 2 c c en 8 b g s 64 Table 11. (continued) Colonial Isolate Cell Pigment Cell Characteristics Number Morphology Color Dimensions 1 2 3 4 5 6 7 55 rod tan .8x1.7-2.9 4 c c en 3 b g 56 rod‘ gray l.2-1.7x4.1-4.5 2 c c en 5 b g 57 rod tan .8x1.7-2.9 2 c c en 3 b g 58 rod gray .8-1.2x1.7-3.7 5 c c en 5 b g 59 rod orange d .8x2.0-3.3 2 c c en 5 b g 60 rod orange d .8x2.0—3.3 3 c c en 3 b g 61 rod tan l.2x2.0-3.7 7 c c en 3 b g 62 rod orange d .8 x 2.5 2 c c en 5 b g 63 rod cream .8x2.0-3.3 2 c c en 3 b g 64 rod pale yel .8-1.2x2.5-3.3 3 c c en 8 b g 65 rod unpig .8 x 2.0 2 c c en 3 b g 66 rod tan 1.2x2.0-2.5 7 c c en 3 b g 67 rod YG d .8-1.2x2.0-2.9 4 c c en 8 b g 68 rod YG d .8x2.5-3.7 3 c c en 5 b g 69 rod white .8x 2.0-2.9 3 c c en 8 b g 70 rod YG d .8x2.5-3.3 3 c c en 3 b g 71 rod white <8-.8x1.7-2.0 4 c c en 8 b g 72 rod YG d .8x1.7-2.5 3 c c en 3 b g 73 rod YG d <8-.8x2.0-4.1 3 c c en 3 b g 74 rod white 1.2x2.5-3.7 5 c c en 3 b g 75 rod YG d .8x1.7-3.3 3 c c en 3 b g 78 rod gray .8 x 2.9 5 c c en 3 b g 79 rod YG d .8x2.9-3.7 4 c c en 3 b g 80 rod white 1.2x2.9-4.1 6 c c en 3 m g 81 rod white 1.2x2.9-3.3 3 c c en 3 b g 82 rod white .8 x 2.5 4 c c en 5 b g 83 rod white .8-1.2x2.0-3.7 2 c c en 3 b g 84 rod YG d .8x2.9-3.3 3 c c en 3 b g 85 rod white .8-1.2x2.0-4.5 3 c c en 3 b g 86 rod cream .8-1.2x2.5 1 c p en 3 b g 87 rod white .8x2.0-2.9 2 c c en 3 b g 89 rod orange .8x2.0-3.7 2 c c en 3 b g 90 rod white .8 x 2.5 1 c c en 3 b g 91 rod white ' -, - - - - - - - 97 rod yellow <8-.8x2.5-3.3 2 c c en ro b g 98 rod gray <8-.8x1.7-2.5 3 c c en 5 b g 99 rod white .8 x 2.5 2 c c en 3 b g 101 rod cream .8x2.5-2.9 1 c c en s b g 102 rod cream 1.2 x 2.5 l c c en 3 b g 103 rod cream .8 x 2.5 2 c c en 5 b g 104 rod cream - 2 c c en 3 b g 105 rod BG d .8x2.0-2.9 7 c c en 3 b g mmmmmmmmmmmmmmmmmmmmmmmmmmmmm mlmmmmmmlmmmm 65 Table 11. (continued) Colonial Isolate Cell Pigment Cell Characteristics Number Morphology Color Dimensions 1 2 3 4 5 6 7 8 106 rod yellow .8x1.7-2.5 p c u u s d g s 107 rod. gray .8x2.0-2.5 3 c c en 3 m g s 881 rod white - p c c en s b g - 108 rod unpig .8x1.7-2.9 l c c u s b g s 110 rod cream d 1.2x2.0-2.9 3 c c en 8 b g s 111 rod gray <8-.8x1.7-2.5 3 c c en 3 b g s 882 rod cream 1.2x2.5-3.7 2 c c u ro b g s 114 rod - .8x1.7-2.5 2 c c en 3 b g s 115 coccus unpig 1.2 p c c en 8 b g d 1181 rod cream .8 x 1.7 l c 0 er s b g s 126 rod cream <8-.8x1.7-2.0 2 c c er s b g s 129 rod cream .8 x 2.0 2 c c en 3 b g s 133 rod cream .8x1.7-2.0 2 c c en 5 b g s 135 rod pale yel .8x1.7-2.0 2 c c en 3 b g s 137 rod yellow 1.2 x 2.5 3 c c en 5 b g s 141 rod cream .8x1.7-2.0 2 c c en 3 b g s 143 rod gray .8 x 2.9 2 c p en 3 m g s 144 rod cream .8x2.0-2.5 2 c c en 3 b g s 145 rod unpig — - - - - - - - - 1471 pleomorph cream - 1 c u er ro b g - 148 rod cream .8 x 2.5 2 c c en 3 b g s 149 rod white .8 x 2.0 4 c c en 3 m g s 151 rod‘ cream .8x2.0-3.3 1 c c en 3 b g s 153 rod pale yel .8 x 3.7 l c c en 3 b g s 154 rod cream .8 x 2.0 5 c r er ro b g s 155 rod white .8-1.2x1.7-3.3 l c c en 3 b g s 156 rod blue d .8 x 2.5 5 c u er ro b g s 162 rod blue d <8-.8x2.5-3.3 5 c u er ro b g s 163 rod white .8x1.7-2.5 4 c p en 5 m g s 164 rod blue d .8x1.7-2.5 5 c u er ro b g s 165 rod blue d <8-.8x2.0-2.5 4 c u er ro b g s 166 rod blue d .8x2.0-2.5 3 i u er ro b g s 167 rod blue d 1.2x2.9-3.7 4 c u er ro b g s 171 rod cream 1.7x3.3-5.0 1 c c u ro b t c 172 rod cream .8 x 2.5 1 c c en 3 b g s 173 rod unpig 1.2x2.9-3.3 2 c p en ru b g s 174 rod cream .8 x 1.7 3 c c en s b g s 66 Table 11. (continued) Colonial Isolate Cell Pigment Cell Characteristics Number Morphology Color Dimensions 1 2 3 4 5 6 7 175 rod yellow .8x4.l-6.6 2 c c u ro b o s 176 rod. yellow .8 x 4.1 2 c c u ro b o s 177 rod yellow .8 x 3.7 3 c c u ro b o s 178 rod yellow .8x3.3-5.8 1 c c u ro b o s 179 rod ' cream .8x2.5-2.9 1 c c en 3 b g s 180 rod yellow .8x2.5-4.l 2 c c u ro b g s 183 rod cream .8x2.0x2.5 5 c c en 5 b d s 184 rod cream .8x2.5- 4.1 4 c c en 8 b d s 185 rod cream .8-1.2x2.9 5 c c en 3 b d s 188 rod cream .8x1.7-2.5 2 c c en 3 b o s 189 rod cream .8 x 3.3 2 c c en 3 b o c 190 rod cream .8x2.5-2.9 2 c c en 5 b o s 191 rod cream 1.2 x 2.9 2 c c en 3 b g s 192 rod cream J- 1. 2x2. 9-3. 3 2 c c en s b g c 193 rod cream .8-1. 2x5. 0- 6. 6 2 c c en 5 b g c 195 rod yellow .8x1. 7- 2.0 2 i c en 3 b g s 196 rod cream .8- 1. 2x2.5-3. 7 2 c c u s b g s 199 rod cream 1.2 x 5.3 2 c c en 3 b g c 202 rod cream .8 x 2.0 3 c c en ru b g c 204 rod white .8 x 2.9 4 10 c f * ro b c s 205 rod cream .8x2.0- 2.9 2 c c en 3 b g s 206 rod cream .8x1.7-2.9 3 i u u s b d s 220 rod yellow .8x2. 0- -2.9 2 c c u s b g s 221 rod yellow .8x2. 9- 4.1 2 c c en 3 b g s 223 rod - .8 x 2.0 1 c c en 3 b g s 224 rod yellow .8 x 2.5 2 c c u ro b g s 2312 rod gray .8 x 3.3 2 c u er ro d g s 232 rod gray .8 x 3.3 3 c c en 3 b g s 233 rod gray .8-1.2x1.7-2.5 3 i c en 3 b g s 234 rod gray .8-1.2x2.0-2.9 4 c c en 3 m g s 991 rod white .8x2.5- 3.7 3 c c er r0 b o s 992 rod orange d .8x3.7- 4.1 4 c c en 5 b g s 993 rod yellow .8x2. 0- 3.3 4 c c u s b o s 994 rod 4 white .8- 1. 2x1.7- 2.0 7 c u er ro b o s 995 rod gray .8x2. 5-4. 1 3 c c en 3 b g s 996 rod blue d .8x1.7-L 5 3 c c er ro b o s 997 coccus cream .8 x 1.2 1 c p en 3 b g s 998 rod yellow .8x2.5-3.3 2 c c u s b g s 999 rod yellow .8x1.7-2.9 3 c c en 3 b g s A 67 Table 12. Names of the Denitrifiers. Isolate Isolate Isolate Isolate Number Name Number Name 4 A. faecalis 61 P. fluorescens II 6 P. type 2 62 P. aureofaciens 12 P. type 2 63 P. fluorescens II 13 P. fluorescens II 64 P. fluorescens II 14 P. type 1 65 A. faecalis 15 P.fluorescens (?) 66 P. fluorescens II 16 P.fluorescens II 67 P. fluorescens II 17 A. faecalis 68 P. fluorescens II 18 A. faecalis 69 P. type 5 19 A. faecalis 70 P. fluorescens II 20 A. faecalis 71 P. type 5 21 A. faecalis 72 P. fluorescens II 22 A. faecalis 73 P. fluorescens II 24 A. faecalis 74 P. type 5 25 A. faecalis 75 P. fluorescens II 26 A. faecalis 78 P. type 6 27 A. faecalis 79 P. fluorescens II 28 A. faecalis 80 P. fluorescens II 29 A. faecalis 81 P. type 2 30 A. faecalis 82 P. type 5 31 A. faecalis 83 P. type 2 36 unknown type 3 84 P. type 6 37 A. faecalis 85 P. type 7 39 unknown type 3 86 A. faecalis 40 A. faecalis 87 P. type 8 41 A. faecalis 89 P. fluorescens II 42 P. fluorescens II 90 A. faecalis 43 A. faecalis 91 A. faecalis 44 P. fluorescens II 97 P. type 9 45 P. fluorescens II 98 P. fluorescens II 46 Flavobacterium sp. 99 A. faecalis 47 P. fluorescens II 101 A. faecalis 48 Corynebacterium sp. 102 A. faecalis 49 P. fluorescens II 103 P. type 10 51 P. type 2 104 A. faecalis 52 P. fluorescens II 105 P. fluorescens II 53 P. fluorescens II 106 A. faecalis 54 P. type 2 107 P. type 11 55 P. type 4 881 A.faecalis 56 P. type 2 108 unknown type 3 57 P. fluorescens II 110 P. type 12 58 P. fluorescens II 111 P. f1uorescens(?) 59 P. aureofaciens 882 P. type 13 60 P. aureofaciens 114 P. type 14 68 Table 12. (continued) Isolate Isolate Isolate Isolate Number Name Number Name 115 unknown type 15 195 P. stutzeri 1181 P. type 16 196 P. f1uorescens(?) 126 P. type 18 199 unknown type 22 129 P. type 18 202 P. type 23 133 P. type 18 204 unknown type 24 135 P. type 18 205 P. fluorescens II 137 P. type 17 206 P. fluorescens IV 141 P. type 16 220 P. stutzeri 143 P. type 11 221 P. stutzeri 144 A. faecalis ‘ 223 A. faecalis 145 P. solanacearum 224 P. stutzeri 1471 Corynebacterium sp. 2312 P. stutzeri 148 A. faecalis 232 P. type 25 149 P. type 11 233 P. type 25 151 P. type 19 234 P. type 11 153 P. type 19 991 P. denitrificans ATCC 13867 154 A. eutrophus 992 P. aureofaciens ATCC 13985 155 P. type 20 993 P. mendocino ATCC 25411 156 P. aeruginosa 994 A. faecalis ATCC 8750 162 P. aeruginosa 995 P. fluorescens II ATCC 17822 163 P. type 11 996 P. aeruginosa 164 P. aeruginosa 997 Pa.denitrificans ATCC 2008 165 P. aeruginosa 998 P. stutzeri ATCC 17588 166 P. aeruginosa 999 P. perfectomarinus 167 P. aeruginosa 171 A. faecalis 172 P. type 16 173 P. type 14 174 P. type 16 175 Flavobacterium sp. 176 Flavobacterium sp. 177 Flavobacterium sp. 178 Flavobacterium sp. 179 P. type 19 180 Flavobacterium sp. 183 P. fluorescens IV 184 P. fluorescens IV 185 P. fluorescens IV 188 P. type 11 189 unknown type 21 190 P. f1uorescens(?) 191 A. faecalis 192 Bacillus sp. 193 Bacillus sp. Table 13. Number and Insertion of Flagella of Selected Denitrifiers. Isolate Number of Flagella Insertion of Flagella 4 l polar or subpolar (based on 1 cell) 17 1 polar 21 1, some 2 polar 43 3 - 4 side attachment 62 l and 2 polar 65 3 - 4 side attachment 107 l polar or subpolar (based on 2 cells) 154 1 polar. 156 1 polar 174 l polar 191 1, some more side attachment Alcaligenes faecalis ATCC 8750 1 side attachment (based on 1 cell) Pseudomonas fluorescens II up to 4 polar ATCC 17822 Pseudomonas perfectomarinus 1 polar or subpolar 70 Table 14. Samples, Contributors, and Their Addresses. Sample Contributors and Their Addresses Minnesota Dr. Robert G. Gast Dept. of Soil Science Univ. of Minnesota St. Paul, Minn. 55101 California Dr. Francis E. Broadbent Dept. of Soils and Plant Nutrition Connecticut 1 and Connecticut 2 Argentina(SP) Argentina(B) Argentina(P) Brazil Michigan(muck), Michigan(Pl), and Michigan(P4) College of Agriculture Davis, Cal. 95616 Dr. James L. Starr Dept. of Soil and Water, Connecticut Agricultural Experiment Station Box 1106 New Haven, Conn. 06504 Ing. Arg. Adolfo Amma INTA Est. Exp. San Pedro Bs.As. Argentina Dr. Ana Garay Departmento de Scielos Escuela de Agronomic INTA - Balcarce Argentina In. Agr. O. Moresco INTA Est. Exp. Parana Entre Rios Argentina Ing. Eli Sidney Lopez Instituto Agronomico Avenido Barao de Itapuro, 1481 Caixa Postal 28 13100 Campinus Est. de Sao Paulo Brasil Dr. James M. Tiedje Dept. of Crop and Soil Science M.S.U. East Lansing, Michigan 48824 71 Table 14. (continued) Sample Contributors and Their Addresses Nigeria(C) and Nigeria(R) Columbia Philippines Taiwan Louisiana Utah Kansas Poultry waste Michigan(WG) Dr. A. Ayanaba International Institute of Tropical Agriculture 0Y0 Road P.M.B. 5320 Ibadan, Nigeria Romeo Martinez CIAT Cali, Columbia Dr. Nyle C. Brady, Director International Rice Research Institute P.O. Box 1300 Makati Commercial Center Makati, Rizal D-708 The Philippines Prof. M. H. Wu Dept. of Soil Science National Chung-Hsiang Univ. Taichung, Taiwan Rep. of China Dr. W. H. Patrick, Jr. Dept.of Agronomy Louisiana State Univ. Baton Rouge, Louisiana 70803 Dr. John J. Skujins Dept. of Biology-UMC 55 Utah State Univ. Logan, Utah 84322 Dr. Larry Murphy Dept. of Agronomy Waters Hall Manhatten, Kansas 66506 Dr. Tata D.S. Prakasam Dept.of Agricultural Engineering Cornell Univ. Ithaca, New York 14850 Dr. Michael Klug Kellogg Biological Station Hickory Corners, Michigan "17111117 11111111111111” ITS