CULTURAL PARAMETERS FOR ESTIMATENG SIMAZiNE EFFECTS ON 883%... MICROBML POPULATEWS Thesis for the Degree 0% M. a 552mm}! STATE UNIVERSITY RAYMOND STEWART SMSTH 1.967 L Ilflluflwmmommmmu 1m: E: R :3 Michigan State University ABSTRACT CULTURAL PARAMETERS FOR ESTIMATING SIMAZINE EFFECTS ON SOIL MICROBIAL POPULATIONS by Raymond Stewart Smith Nitrogen sources, growth factors and incubation atmos— pheres were investigated as cultural parameters which might be useful in dilution plating procedures to detect changes in nitrate reductase activity in soil microbial populations. Criteria were sought which might differentiate between'bac— terial types in which nitrate reduction is (l) essentially assimilatory and those in which it is (2) essentially or (3) coincidentally dissimilatory. Simazine, a herbicide known to stimulate nitrate re- ductase activity in higher plants, was tested for its ability to promote the same adaptive response in 18 strains of known facultative nitrate reducing bacteria. The observed re- sponses of these strains in pure culture were used as the basis for inferences regarding population estimates obtained under parallel cultural conditions for field soils previously treated with simazine. Simazine introduced into solid media at concentrations of 5 to 50 ppm promoted more rapid development of streak cultures of several nitrate reducing bacteria. This stimulus Raymond Stewart Smith to growth appeared to be due to earlier adaptive synthesis of nitrate reductase, since there were no effects of Simazine on growth or denitrification when added to broth cultures pre- viously adapted to nitrate. Field Simazine treatments reduced plate counts for major taxonomic groups in the order: bacteria (66%) > actino mycetes (25%) > fungi (15%). The proportion of nitrate re- ducers in the bacterial population, however, was markedly in- creased, as evidenced by numbers capable of growth and/or sur- vival in 100 per cent CO2 or H2 on media supplying nitrate. The major proportionate increase in nitrate reducing bacteria occurred among types which differed from represen- tative pure strains of denitrifying bacteria in their ability to grow on nitrate media only after removal from CO2 into air. It is inferred that these are not actively denitrifying types, but rather types in which nitrate reductase activity, stimu- lated by Simazine, is exploited for assimilitory reduction of nitrate to support growth. . It appeared that the differentiation between assimila- tory and dissimilatory nitrate reducers could be clearly made using dilution plating techniques on solid media. The cul— tural parameters employed with solid media did not provide criteria for the clear distinction between true denitrifiers and coincidental dissimilatory nitrate reducers which is possible with broth media. CULTURAL PARAMETERS FOR ESTIMATING SIMAZINE EFFECTS ON SOIL MICROBIAL POPULATIONS BY Raymond Stewart Smith A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Soil Science 1967 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. A. R. Wolcott for his assistance and encouragement throughout this study. The author wishes to thank all those members of the faculty of Michigan State University whose cooperation has made this study possible. The author is also grateful to his wife, Cynthia, for her unfailing support, help and encouragement during the course of this work. The study was supported by Public Health Service re- search grant CC 00 246, from the National Communicable Di- sease Center, Atlanta, Georgia. ii TABLE OF CONTENTS Page INTRODUCTION 0 o o o o o o o o o o o o o o o o o o o o l LITERATIJRE REVIEW 0 o o o o o o o o o o o o o o o o O 3 EXPERIMENTAL METHODS . . . . . . . . . . . . . . . . . 28 Nitrate Reducing Bacteria - Pure Culture Studies . 28 [Microbial Counts from Simazine Treated Plots . . . 34 Denitrification in Liquid Broth . . . . . . . . . 35 RE SULT S O O O O O O O O O O O O O O O O O O O O O O O 38 Cultural Studies on Solid Media . . . . . . . . . 38 Responses to inositol and thioglycollate . . . 39 Interactions of nutrition, atmospheric composition and Simazine on growth . . . . . 43 Interactions of nutrition, atmospheric composition and Simazine on antagonism by_§§. aeruginosa . . . . . . . . . . . . . 49 Interaction of nutrition, atmospheric composition and Simazine on pigment pro- duction by_g§. aeruginosa . . . . . . . . . 55 Population Studies with Soils Treated in the Field with Simazine Plus Amitrole-T . . . . . . 59 Effects of Nutritional Parameters and Simazine on Growth and Denitrification in Broth Media . . 68 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 78 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 82 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 85 iii LIST OF TABLES Table Page 1. Nitrate reducing bacteria investigated in pure culture . . . . . . . . . . . . . . . . 29 2. Experimental media used to supply growth factors and sources of carbon and nitrogen for cultural studies . . . . . . . . . . . . 31 3. Aerobic growth on agar media at 28C . . . . . 40 4. Anaerobic growth on agar media at 28C . . . . 42 5. Growth of denitrifying bacteria on solid media in various atmospheres . . . . . . . . 45 6. Growth inhibition and stimulation of various denitrifying bacteria by Pseudomonas aeruginosa (ATCC 10145) under various atmospheres on cross-streaked agar plates . 54 7. Inhibition by various denitrifying bacteria of pigmentation in Pseudomonas aeruginosa (ATCC 10145) on solid media under H2 atmosphere 0 O O O O O O O O O O O O I O O O 57 8. Microbial population estimates and statisti- cal probabilities for differences between controls and soils with previous history of field treatment with simazine plus Amitrole-T . . . . . . . . . . . . . . . . . 62 9. Geometric mean numbers and relative numbers of microbial groups in control soils and soils with previous history of field treat— ment with simazine plus amitrole-T . . . . . 64 10. Cultural responses of denitrifying bacteria in medium 103 broth with and without SimaZine O O O O O O O O O O O O O O O O O O 70 11. Cultural responses of denitrifying bacteria in medium 105 broth with and without simazine . 71 iv Table Page 12. Cultural responses of denitrifying bacteria in medium 106 broth with and without simazine . . . . . . . . . . . . . . . . . . 72 13. Cultural responses of denitrifying bacteria in medium 113 broth with and without simazine . . . . . . . . . . . . . . . . . . 73 Figure LIST OF FIGURES Effect of amino acids on utilization of glucose and fermentation products by Pseudomonas stutzeri for aerobic growth with N supplied as (NH4)2SO4 . . . . . . . Effect of amino acids on utilization of tricarboxylic acid cycle components and associated metabolites by Pseudomonas stutzeri for aerobic growth with N sup- plied as (NH4)ZSO4 . . . . . . . . . . . . Interactions of amino acids and simazine on aerobic growth in glucose-nitrate broth at 30C of (a) Pseudomonas aeruginosa, (b) _g§. stutzeri, and (c) Serratia marcescens Interactions of amino acids and simazine on aerobic growth in glucose—nitrate broth of Pseudomonas aeruginosa at 10 and 20C . . . Aerobic growth and interactions of_g§. aeruginosa (strain 1) and B. licheniformis strains 6, 8 and 14 . . . . . . . . . . . Aerobic growth and interactions of_g§. aeruginosa strain 1 and g. laterosporus [5], B. coagulans [7] and-g. cereus [9] Growth and interactions in C02 of_g§. aeruginosa strains 1 and 2,_§§. stutzeri [3] and_g§. fluorescens [l7] . . . . . . . Growth and interactions in C02 of_g§. aeruginosa strain 1 and g. laterosporus [5], B. coagulans [7] and_§. cereus [9] . Growth and interactions of Ps. aeruginosa strain 1 and_§. licheniformis strains 6, 8 and 14 in a hydrogen atmosphere . . . . . vi Page 12-A 13-A 26-A 26-B 52-A 52-A 52—B 53-A Figure Page 10. Growth and interactions of_g§. aeruginosa strain 1 and_§. licheniformis strains 6, 8 and 14 after 2 days in air following 8 days in hydrogen . . . . . . . . . . . . O 53-A 11. Effects of field treatment with simazine plus amitrole-T on the distribution of cultural groups in the bacterial popu- lation defined by aerobic plate counts on medium III . . . . . . . . . . . . . vii INTRODUCTION Soil microbial responses to pesticides are generally evaluated by monitoring microbial metabolism (CO2 evolution, oxygen uptake, ammonification, nitrification, cellulose de— composition, etc.), or by estimating numbers of the major taxonomic groups (bacteria, actinomycetes and fungi). These methods obtain no information on the variances within taxo- nomic or physiological groups. Certain physiologically simi- lar species may find the induced conditions detrimental to survival while others are able to increase in numbers, thus the opposite effects balance and portray a picture of little or no change. Although ideally each microbial specie would be investigated, this is obviously impossible. A compromise approach is to classify the population into increasingly re- strictive physiological groups and develop appropriate methods for observing variations at these levels. Denitrifying bacteria include genera and Species which, under anaerobic conditions, reduce nitrate to gases which are lost from the soil. An adaptive mechanism neces- sary for this dissimilatory reduction is the synthesis of nitrate reductase. Recent studies have shown that simazine, an agricultural herbicide, stimulates nitrate reductase activity in higher plants. Were this response to occur in the denitrifying bacteria, an enrichment in denitrifying ca- pacity would develop in the soil population. Enhancement of nitrate reductase activity in the soil microflora need not necessarily increase denitrification losses. Depending upon the balance of responding organisms, an increase in nitrate reductase activity in the soil mi- crobial population could lead to increased losses of nitrogen due to denitrification, or to decreased losses due to re- duction to nitrite, ammonium or to amino N in cellular tissues. Present methods for estimating numbers of denitrifiers in soil populations do not permit distinctions to be made be- tween these different types of nitrate reducing organisms. These methods, involving serial dilutions in broth culture and plus or minus observations on growth or production of gas and alkalinity, must be handled by statistically weak "most proba- ble numbers" techniques. The main objective of the present study was to investi- gate cultural parameters which might permit use of dilution plate counts on solid media to detect changes in nitrate re— ductase activity in soil populations and to differentiate be— tween important types of nitrate reducing organisms. Effects of nutritional and atmospheric parameters were tested in pure culture with and without simazine. Se- lected combinations of media and incubation atmospheres were used to estimate population changes in field soil previously treated with simazine. LITERATURE REVIEW Denitrification The loss of gaseous nitrogen from soils may occur under various environmental conditions with several resulting products. Four distinct mechanisms are suggested by Alexander (1): a) an-biological losses of ammonia which, according to Willis and Sturgis (109), may be significant only above pH 7.0. b) Chemical decomposition of nitrite under acid con- ditions to yield nitrogen oxides. Clark, Beard and Smith (20) suggest reduction of nitrite to nitric oxide as the main mechanism for nitrogen loss in well—aerated acid soils. Wullstein and Gilmour (112) have presented evidence that transition metals may catalyze these reactions. C) Production of N2 by the non-enzymatic reaction of nitrous acid with ammonium or amino acids. Bremner (9), Mortland and wolcott (66), and Stevenson and Swaby (89) conr sider it quite likely that non-enzymatic reactions of nitrous acid or its equilibrium species with phenolic structures in humus or decomposing biological materials are significant pathways for loss of gaseous nitrogen under field conditions. d) Enzymatic denitrification, which is a biological pro- cess accomplished by facultatively anaerobic bacteria capable of using nitrate or nitrite in place of oxygen as a hydrogen acceptor. This dissimilatory reduction is in contrast with the assimilatory reduction of nitrate or nitrite to the level of ammonium or amino nitrogen, as accomplished by higher plants and many microorganisms to meet their requirements for organic nitrogen. Considerable research has been directed toward the investigation of the pathways for denitrification. There is fairly general agreement that the reduction sequence includes nitrate, nitrite, nitrous oxide, and molecular nitrogen, in that order. However there is still some disagreement regard- ing the inclusion of nitrous oxide on the main pathway of denitrification. Allen and Van Niel (4) concluded that nitrous oxide was not the precursor of molecular nitrogen in cultures of Pseudomonas stutzeri. Wijler and Delwiche (108) found nitrous oxide to be the major denitrification product under moist soil conditions. They noted, as did Hauck and Melsted (39), that above pH 7.0 nitrous oxide could be readily re- duced to nitrogen, but below pH 6.0 its reduction is strongly inhibited. On the other hand Delwiche (26) found that the relative proportions of N20 and N2 were markedly influenced by the concentration of nitrate plus nitrite. Because ni- trate and nitrite are preferential hydrogen acceptors N20 is utilized, and subsequently reduced to N2, only if the supply of nitrate and nitrite is limiting. However, most of the published evidence is in agreement with memik's (69) con- clusion that nitrous oxide is an obligatory precursor of molecular nitrogen. Several soil environmental factors have a direct in- fluence on denitrification. There is general agreement among several investigators, Valera and Alexander (99), Delwiche (25), and NOmmik (69), that microbial denitrification is only of consequence in near-neutral soil habitats (pH 6.0 to 8.0). Broadbent and Clark (11) stated that the effect of organic matter on denitrification is twofold: since the free energy change in the reduction of nitrate to nitrous ox- ide or molecular nitrogen is positive, an oxidizable sub- strate is required which can furnish energy for growth of the denitrifying bacteria and serve as a hydrogen donor for the denitrification process; and secondly, the rate of organic matter decomposition markedly influences the oxygen demand. The firSt effect is illustrated by McGarity (62) who showed that the addition of glucose markedly increased the rate of denitrification in soil with low content of organic carbon, but the increase was only slight in soils with high native levels of organic carbon. Denitrification is markedly affected by temperature. The optimum is above 25C. memik (69) and Bremner and Shaw (10) reported an optimum as high as 60-65C. Although the re— action is decreased at lower temperatures, Alexander (1) con- siders it still to be of economic importance. Moisture level and oxygen availability, which are in an inverse relationship in the soil, are two final environ- mental factors which influence denitrification. Moisture level has a direct effect on denitrification as reported by Jansson and Clark (40), NOmmik (69), and Bremner and Shaw (10). The latter authors observed increased nitrogen losses as a function of moisture content up to 450% water-holding capacity. More recently Mahendrappa and Smith (59) have re- ported the fastest nitrogen gas production under anaerobic conditions at moisture near saturation, with a reduction in rate at levels above and below this point. The oxygen supply influences denitrification signifi- cantly, for the utilization of a nitrogen oxide as a hydrogen acceptor will occur only when the oxygen supply is insuffi- cient. Mbst investigators, including Allison and Carter (5), Bremner and Shaw (10), and Alexander (1), conclude that de— nitrification may continue in anaerobic microhabitats of a well aerated soil, but that the nitrogen loss by this bio— logical mechanism is not significant under these conditions. Several investigators, Broadbent and Stojanovic (12), Kefauver and Allison (47), Marshall, Dishburger: MacVigar and Hallmark (60), Meiklejohn (63), Treccani (97), and Verhoeven (101), have reported aerobic denitrification, but have not estimated the oxygen levels during their investigation. In contrast Cady and Bartholomew (17) reported an atmospheric oxygen level less than.7% by volume necessary for appreciable denitrification, while Greenwood (34), and Sher- man and MacRae (86) consider denitrification significant only at dissolved oxygen levels below 0.2 ppm in the soil solution. Differing from the above views, which regard oxygen deficiency as imposing a reliance upon oxidized forms of nitrogen as alternative electron acceptors, Pichinoty (73) and Chang and Morris (18) propose that oxygen inhibits bac— terial denitrification by repressing the biosynthesis of ni- trate, nitrite, and nitrous oxide reductases. They suggest that oxygen also inhibits the action of these reductases after they are formed. Denitrifying Bacteria Many bacterial species have been shown to denitrify, in culture media at least, and large populations of several of the more active bacteria are found in most agricultural soils. Wbldendorp (111) has found that non—spore—forming organisms of the genera Pseudomonas, Micrococcus and Spirillum, aerobic spore formers (Bacilli) and a number of other facul- tative anaerobes can reduce nitrate. The active species are largely limited to the genera Pseudomonas, Achromabacter and Micrococcus. Bacillus strains, though numerous, are rarely important because their abundance is usually only the result of the persistence of the endospores (Alexander, 1). Delwiche (25) reports more than forty organisms with the ability to denitrify. From the above summary of denitrifying bacteria, a few physiological similarities are immediately apparent. In order to classify these organisms physiologically and to more fully understand their relationships with plants and other important soil organisms, however, further nutritional and environmental requirements and ecological roles of the de- nitrifying bacteria must be determined. The known denitrifying bacteria may be referred to as chemoorganoheterotrophs, that is they depend upon the oxi- dation of exogenous organic substances for an energy source and may also require an exogenous supply of one or more es- sential metabolites or growth factors. It seems that all bacteria require a small number of nutrients which may be re- ferred to as universally required foodstuffs (Lamanna and Mallette, 51). These nutrients are water, phosphate, carbon dioxide, and certain mineral salts. The differences among bacteria in the foodstuffs required are rarely due to differ- ences in the need for particular elements. Important differ- ences do appear in the compounds of carbon, nitrogen, and sulfur which they can assimilate. Ecological investigations of the N2-releasing bac- teria of soil are frequently performed using Giltay's medium, in which nitrate and minerals are supplied, with citrate and asparagine as carbon sources. Valera and Alexander (100) sug- gest that the energy source selected for denitrification population estimates is a critical factor. They reported that glycerol and tartrate appeared to yield greater popu- lation estimates than the citrate supplied in Giltay's medium. Mbreover, the inclusion of yeast extract in the citrate-inorganic salts medium resulted in an eight-fold greater estimate of the numbers of denitrifying bacteria than when Giltay's medium was used. In parallel pure culture studies these authors also concluded that maltose, xylan, mannitol, lactose, xylose, glucose, raffinose and sucrose could serve as carbon sources for most of the denitrifying species examined. The heterotrophic microflora in soils comprise a very numerous diversity of genera and species, the functional roles of these and their ecological implications are still largely unknown or but little understood. With a view to un- covering some of these functional relationships, efforts have been made to classify soil microorganisms on the basis of nutritional needs. Lochhead and Chase (56) described a classification system based on a determination of growth re- quirements of soil bacteria first isolated on nonselective plating media. Seven main nutritional groups were recognized, ranging from organisms capable of maximum development in a simple basal medium to types unable to develop with supple- ments of amino acids, growth factors, or yeast extract, but which require soil extract for growth: 10 Medium B Basal medium ” A " plus amino acids " G ” plus growth factors (vitamins) ” AG ” plus amino acids plus growth factors ” Y ” plus yeast extract " S " plus soil extract " YS " plus yeast extract plus soil extract Valera and Alexander (100) studied pure cultures of denitrifying bacteria to ascertain some of their nutritional relationships. They concluded that the N2-producing micro— organisms with which they worked could be divided into four nutritional groups: a) Bacteria that denitrify in a glucose—inorganic salts medium, a group that included three Pseudomonas aeruginosa strains and_g§. denitrificans. b) Microorganisms like Achromobacter hartlebii which re— quire ammonium for denitrification. c) Those apparently needing amino acids for activity, Alcaligenes denitrifigans,_g§. stutzeri and Micrococcus-ge— nitrificans being included in this category. d) A fourth group containing Bacillus licheniformis, Denitrobacillus licheniformis, Serratia indica and §. kilensis, none of which denitrified in the test media. Wbldendorp (111), using broth cultures, found that his collection of denitrifying bacteria could be divided into two 11 groups on the basis of whether they could or could not grow anaerobically in the absence of nitrate. Additional dis— tinctions could be made within each of these groups on the basis of whether or not amino acids or vitamins were re- quired for growth and/or gas production. His observations were, in general, consistent with those of Valera and Alex— ander (100). In a comparative survey of the nutrition of the genus Bacillus, Knight and Proom (50) observed the following charac- teristic nutritional patterns: 1. .E- subtilis, B. licheniformis and B. megatherium grew with gluccose as energy source, ammonium as sole nitrogen source and in the absence of added growth factors. 2. 'g. Cereus and_§. brevis grew in the absence of added growth factors but required mixtures of amino acids instead of ammonia only as sources of nitrogen. 3. .g. pumilus and g. macerans grew with ammonia only in the presence of biotin and aneurin. 4. .g..aly§i required amino acids and aneurin; §-.Ei£22‘ _l§n§ and.§. coagulans had more complex requirements. Investigators working with M, denitrificans have ob- served some physiological characteristics differing from the other known denitrifying bacteria. Kluyver and Verhoeven (48) have observed an adaption in M. denitrificans which per- mits chemolithotrophic oxidation of hydrogen at the expense of nitrate. Pichinoty (72) reports two nitrate—reductase 12 enzymes, A and B, in a strain of_M. denitrificans. B is a constitutive enzyme without known physiological functions, whereas A is induced by the presence of nitrate but suppressed by oxygen. A third unique characteristic was observed by Mutze (67) who found that exposure to light decreased the oxidative activity of M. denitrificans due to inactivation of cells which do not possess carotenoids to absorb light of in- jurious shorter wavelengths. It is apparent from the above studies that denitrify- ing organisms represent a wide diversity of nutritional re— quirements with respect to carbon source, energy source, growth factor requirements and the extent to which dissimila- tory nitrate reduction is essential for or incidental to growth under anaerobic conditions. In preliminary work leading to the present study, B. S. Hong1 investigated the effect of amino acids on the abili- ty of Pseudomonas stutzeri to utilize different carbon sources for growth in vigorously aerated broth cultures. Cells were grown into the exponential phase in nutrient broth. Washed cells were used to inoculate test media to a standardized optical density of .01 or .02 at 600 mp, and growth was fol- lowed turbidimetrically at this wavelength. Data in Figure 1 show that only acetate could be uti- lized for maximum growth in the absence of exogenous amino acids. A major deficiency appeared to be for amino acids 1Unpublished data, 1965. 12-A Amoma .mcom .m .mv .vommfiemzv mm 66a .11 .Hmmsm 2 a3 SDBOHm UHQOme How Humnuspm mmGOEOUswwm kn muospoum comwmu IGoEHmm paw mmoosam Mo coapmuflaflus so moaom OCHEm mo “Uummm maze: I us: on ov om ON 0. _ _ — a _ _ _ _ _ 4 28222378 .222... . 383.5 I I I l I I I I II I l \'|"'l""'|"‘\ > \\\ \ _Osoo_0IIII\ _o=o~IIIIIIIIII \\\. _O=oqcbm I I I I I I ISIUI IIIIIII I I HIIII\“\\ 222$IIIIIIII\\ 8.04.59 53 um I I I I me: 0254 oz :3. 28:323. IX! .22.; a 2:: 3.38:. £28 bacteria > fungi. Mbst organisms recovered when exposed to air, indicating that organisms were not killed, only growth was inhibited. Es— sentially no organisms were capable of growing under 100 per cent carbon dioxide. The inhibition of most microorganisms by high concen— trations of carbon dioxide was not caused by a deficiency of oxygen, inasmuch as a higher percentage of organisms were capable of developing under 100 per cent nitrogen than under carbon dioxide tensions greater than 90 per cent. In a recent study Stotzky and Goos (91) confirmed pre— vious observations (90,92) that the soil microbiota can adapt to conditions of poor aeration. The increase in numbers of microorganisms tolerant to high tensions of carbon dioxide and low tensions of oxygen after conditioning of soil to these conditions were similar to the higher numbers of carbon di- oxide tolerant organisms counted in soils stored for three months in plastic bags than from soils analyzed immediately 23 after sampling in the field. It could not be conclusively established whether conditioning resulted in enrichment of specific groups within the soil population which were al- ready tolerant to these conditions, or whether physiological adaptation occurred. Simazine Effects Simazine (2-chloro-4,6—bis(ethylamino)—S—triazine) is a herbicide used for selective weed control on several crOps. When used at the recommended field application rate, from 1 to 4 lbs/acre, Alexander (3) and Talbert and Fletchell (94) have reported the persistence of simazine in the soil up to 16 months. Ercegovich (29) has reviewed the literature con- cerning the mechanisms for disappearance of simazine activity. These include: (a) volatilization, (b) adsorption by the soil colloidal complex, (c) leaching, (d) chemical alteration, (e) photodecomposition, (f) plant removal, and (g) microbial degradation. Numerous investigators, including Audus (6), Burnside .§E_El‘ (14), Kaufman_g£_al. (46) and Guillemat (36) have re— ported fungi and actinomycetes isolated from soil which were capable of degrading simazine and which, in the process, uti- lized simazine as nearly the sole source of carbon and/or nitrogen. However, Pantos et_gl. (70) and Farmer §t_al. (30) included, also, a few bacterial species which were capa- ble of degrading simazine. 24 It is agreed that the microbial population affects simazine. Conclusions from research into the reverse situ- ation, the effects of simazine on the microbial population, do not receive such general agreement. Several investigators, including Burnside gt_§l. (l4), Eno (28), Guillemat_gt_§1. (37), Pantos §t_al. (70), Stein- brenner_§E_§l. (88), Todorovié and Grbié (96), and volk and Eno (102) studied the effects of simazine on the general microbial population. They observed CO2 production and/or bacterial, fungal and actinomycete numbers. No significant effects were detected when simazine was applied at recommended field rates. However, Ragob and McCallum (74), after treat- 14 ing a soil with C labeled simazine, reported a drop in 14CO2 produced after 91 hours, indicating simazine inhibition. Similar conclusions were drawn by Klyuchnikov et al. (49L who observed a decrease in bacteria, fungi and actinomycetes with the recommended rate applied to a sandy soil. Nepomiluev_gt '31. (68) suggested that the lower initial microbial popu- lation and the greater inhibitory effect of simazine in a sandy soil, compared with a soil which had received high rates of organic matter and fertilizer, were due to the low level of nutrients. The effect of simazine on the bacteria involved in nitrogen transformations has been a concern to several in— vestigators. No significant effects of simazine on nitrifi- cation in soil systems were reported by Burnside et a1. (14) for dosages up to 4096 ppm, by Eno (28) up to 16 pounds per 25 acre, or by Volk and Eno (102) at recommended field rates. On the other hand Tsvelkova (98) obtained data following the application of simazine which indicated a 10-fold increase in nitrification in a soil with pH 5.3 and 2 per cent organic matter. In a perfusion study, Farmer_et_al. (30) found an in- hibition of nitrification above 6.0 ppm of simazine. They concluded from pure culture studies that this reaction was stopped by the inhibition by simazine of Nitrobacter and that Nitrosomonas was not inhibited. Since the solubility of simazine in water is only 5 ppm, Volk and Eno (102) suggest that this is not enough to affect bacterial growth, but that phenomena associated with physical contact of the microbes with simazine particles be- comes important in altering growth. Perhaps the simazine enters the organisms by virtue of the fact that it is soluble in components of cell walls or cytoplasmic membranes. No reports of simazine effects on nitrogen minerali- zation and denitrification are available. It may be specu- lated from observations of Nepomiluev et al. (68) that indi- vidual microbial species may vary greatly in their suscepti- bility. These investigators found that non-spore-forming bacteria were more resistant to simazine than fungi and actinomycetes, and much more resistant than spore—forming bacteria. 26 Hong3 conducted preliminary tests with simazine to evaluate its effect on growth of three non—spore—forming bacteria which are known denitrifiers: _P§. aeruginosa,_g§. stutzeri and §. marcescens. In vigorously aerated (roll- tube) cultures in glucose-nitrate broth, simazine at concen— trations of .005, .5 or 3 ppm had no effect on growthat 30C when amino acids were present (Figure 3). In the absence of growth factors, simazine at 3 ppm reduced the lag period for all three organisms and materially increased the rate of growth of P3. aeruginosa and §. marcescens. Tests at lower temperatures were conducted with.g§. aeruginosa only (Figure 4). In the presence or absence of growth factors, lag periods were increased by 3 ppm simazine. At 20C, subsequent growth was more rapid in the presence of simazine. At 10C, the pattern of growth in the presence of amino acids was altered by simazine. In the absence of amino acids, growth at 10C was inhibited by simazine for 180 hours, and very meager development was observed up to 300 hours. Tests by Hong under anaerobic conditions were inconclusive. It has been well established that simazine increases the growth and nitrogen content of tolerant plant species (Bartley, 7; Freney, 32; Karnatz, 41; Reis and Gast, 75; and Reis et al. 76). This effect is not due to a lack ofvnuai 3B. S. Hong, Unpublished data, 1966. 26-A 563 6:21 .m .m: .mcoomoonma maumnuwm ADV paw .Huonusum .wm AQV .mmocstnmm mmc080©5mmm Amy mo Uom um nuonn mumnuaslwmoosam ca nuzonm canonwm co mcHNmEflm paw mpflom ocHEm mo mcofluomumucH .m .mHm mane: om on ow on Imi $3.53 2.3.”: 8.: 2:3 oz we”: 02:: oz I I I 52.22; 2.: m “L we: 02.24 mo. AllSNBO 1V0|ld0 oo._ 8N L 26-B I363 .mcom .m .m: .oom 8cm OH um mmocflmsnom mmeEOpsomm mo suoufl mumanCImmoosHm cfl npzosm UHQOme co wcHNmEHm Ucm mpflom OCHEm wo mCOHuomHmch .v .mam com com on. co. on 0 do. .2 .3: cc om ON 0. .u om .2 .mm: i _o. .. \ .. \ \ -mo. ..\ ..\ \ Inc. 0 \.. M . m o. .I 0 . 3 .8 m U IA .. . . .. Ion. so. . .. .. ......x ...... ozfizza zoom: \ .... x 854 2:: oz \\.oo~I.oI~I. \ \ III 8.2 ozzzz oz Ioo._ do. soul I I \ ..... ...mznsza z& T; 834 2.3 . I I I I 8.2 3:: Too N 27 competition (32,75,76), nor to the additional nitrogen avail— able in simazine (75). In experiments with corn (gga gays L.), Reis and Tweedy (77) found these responses to simazine occur in plants grown with nitrate, but not in plants grown with ammonium as the source of nitrogen, and are greatest when nitrate and temperature are at sub-optimal levels. They also reported nitrate reductase activity in corn growing on sub-optimal levels of nitrate increases in a linear fashion with simazine concentration. From these observations they presented the hypothesis that simazine enhanced nitrate utilization by in- creasing nitrate reductase activity. EXPERIMENTAL METHODS Nitrate Reducing Bacteria - Pure Culture Studies In the review of literature it was observed that ni- trate—reducing bacteria were found in increased numbers in the soil rhizosphere (45,81,85). One of the causes of this ecological phenomenon has been speculated to be the increased supply of amino acids in the rhizosphere (81), but the nu— tritional and/or antagonistic interactions producing this in— crease in denitrifiers have not been described in any detail. A series of experiments were conducted in petri dishes, using pure cultures of known nitrate-reducing bac— teria. The objectives were to find differences in nutritional requirements of representative species which might be used to develop solid media for enumeration and isolation of more re- stricted physiological types among the total group of nitrate reducers, and to observe interactions between the test species on such media under the influence of various atmospheric con- ditions. Simazine was included in some instances to determine its effect on the test organisms. Eighteen species and strains offacultatively anaerobic bacteria with known capacity for reducing nitrate were used (Table 1). Stock cultures were maintained on nutrient agar 28 29 Table l. Nitrate reducing bacteria investigated in pure culture. Laboratory Organism Source Number Identification 15 Achromobacter hartlebii ATCCa 365 9 Bacillus cereus ATCC 6464 10 Bacillus cereus ATCC 14579 13 Bacillus circulans ATCC 4513 Bacillus coagulans woldendorpb 1963 II Bacillus laterosporus WOldendorp 468B Bacillus licheniformis woldendorp Pl 6 Bacillus licheniformis woldendorp 430 14 Bacillus licheniformis ATCC 14580 11 Bacillus macerans ATCC 843 12 Bacillus macerans ATCC 8244 16 Micrococcus denitrificans ATCC 13543 1 Pseudomonas aeruginosa ATCC 10145 2 Pseudomonas aeruginosa woldendorp 18 Pseudomonas denitrificans ATCC 13867 17 Pseudomonas fluorescens ATCC 11250 3 Pseudomonas stutzeri woldendorp ‘§erratia marcescens MSU MPHc aAmerican Type Culture Collection, Rockville, Maryland. bJ.‘W. WOldendorp, Laboratory of Microbiology, Agri- cultural University, Wageningen, Netherlands. cDepartment of Microbiology, Michigan State University. 30 plus yeast extract, with periodic transfer at 30C followed by storage at 10C after cultures reached log phase. Eleven experimental media were employed (Table 2). Media 103 through 115 are modifications of media described by woldendorp (111) and by Valera and Alexander (100). Stock solutions listed in Table 2, when combined in the proportions shown, give the following concentrations of minerals in 1 liter of the final medium: 123 mg NazHPO 4, 724 mg KH2P04, 89 mg MgSO4'7H20, 53 mg CaC12'2H20, 380 ug CuSO4'5H20, 440 ug ZnSO4’7H20, 310 ug MnSO4‘H20, 250 ug H3BO4, 5 ug NaMoO4°2H20 and 1 mg Fe (as FeEDTA) per liter. The media were prepared by combining the buffer so- lutions (in proportions to provide media of pH 7.3), mineral salts solutions and agar in a suction flask. These were sterilized by autoclaving at 121C for 15 minutes. The glu- cose, amino acids, vitamins, iron chelate, and simazine were sterilized by introduction through a Millipore filter. The simazine was added as a concentrated solution in chloroform. The chloroform was removed by aspirator suction in a water bath at 45C. Similar quantities of chloroform were added to and evaporated from control media without simazine. The media were poured into disposable petri plates, cooled and those plates designated for anaerobic incubation were placed immediately into an H2 atmosphere for storage un— til ready for use. Plates for aerobic incubation were stored on the laboratory table under sterilized cloth cover. 31 Table 2. Experimental media.used to supply growth factors w Medium Number Component 103 105 106 107 Stock sol'n. Aa 510 m1 510 m1 510 ml 510 m1 " " Bb 90 ml 90 ml 90 ml 90 ml " " cC 180 ml 180 m1 180 m1 180 ml " " Dd 10 ml 10 ml 10 ml 10 m1 " " Be 10 ml 10 ml 10 ml 10 ml KNO3 2.5 g 2.5 g --- 2.5 g Asparagine --— ——— 2,5 g --- Glucose 2.5 g 2.5 g 2.5 g 2.5 g Vitaminsf -—— 20 ml 20 ml --- Amino acids9 --- 20 ml 20 ml --- Na thioglycollate -—- ——— —_- 500 mg Yeast extract --- -—_ --- --- Na Citrate -—- --- --- --- KH2P04 --- ——— __— --- MgSO4-7H20 -—- ——- --- --- CaC12'6H20 —-— --- --- ___ FeCl3'6H20 —-— --- --- --- Agar 15 g 15 g 15 g 15 g H20 to: 1 liter 1 liter 1 liter 1 liter pH 7.3 7.3 7.3 7.3 aStock sol'n. A: 1.42 9 Na HPO4 per liter (.01-M). 2 bStock sol'n. B: 1.36 g KH2PO4 per liter (.01 M). cStock sol'n. C: 493 mg MgSO '7H20 (.002 M) plus 294 mg CaC12°2H20 (.002_M) per liter. 4 dStock sol'n. D: 38 mg CuSO4-5H O, 44 mg ZnSO4°7H20, 31 mg MnSO4°H20, 25 mg H3B03, and 5 mg Na2M004-2H20 per liter. eStock sol'n. E: 769 mg Fe EDTA per liter. 32 I . a and sources of carbon and nitrogen for cultural studies. Medium Number 109 110 111 112 113 114 115 510 ml 510 ml 510 ml 510 ml -—- 510 m1 510 ml 90 ml 90 ml 90 ml 90 ml --- 90 ml 90 ml 180 ml 180 ml 180 ml 180 ml ——— 180 ml 180 ml 10 ml 10 ml 10 ml 10 ml ——— 10 ml 10 ml 10 ml 10 ml 10 ml 10 ml --- 10 ml 10 ml 2.5 g -—- 1.25 g 1.25 g 1.0 g 2.5 g 2.5 g —-- 2.5 g 1.0 g 1.0 g 1.0 g --- -—- 2.5 g 2.5 g 2.5 g 2.5 g —-- 2.5 g 2.5 g 20 ml 20 ml 20 m1 --- --— 20 m1h 20 mlh 20 ml 20 ml 20 m1 —-- --- 20 ml 20 m1 500 mg 500 mg 500 mg 500 mg 500 mg 500 mg -—- --- --- --- 10 g --- ___ ___ —-- -—— -—— ——— 8.5 g --— -—— --- ——— -—— --- 1.0 g --- --— --- --- --— -—— 1.0 g ——— ——— --- --- ——- ——- 0.2 g --- --- --- --— --- -—- 0.03 g --- --— 15 g 15 g 15 g 15 g .15 g 15 g 15 g 1 liter 1 liter 1 liter 1 liter 1 liter 1 liter 1 liter 7.3 7.3 7.3 7.3 7.3 7.3 7.3 fVitamin stock solution to provide 1 ug biotin, 2 ug vitamin B , 2 ug folic acid, 100 ug riboflavin, 500 ug thia- mine, 500 ug nicotinic acid, 500 ug pyridoxine-HCl, 500 ug ca-pantothenate and 50 mg inositol per liter in the final medium. 9Amino acid stock solution to provide 950 mg vitamin- free casamino acids, 50 mg tryptophane and 10 mg cysteine per liter of final medium. hVitamin stock solution described above without inositol. 33 Inoculating cultures were prepared by growing in nutrient broth for 12 to 18 hours. To prevent a carry over of nutrients, the cells were spun down, the nutrient broth removed and the cells resuspended in buffer solutions A and B (Table 2) combined to provide a pH of 7.3. For anaerobic incubations, atmospheres of H2 and/or CO2 were used in two cabinet type anaerobic incubators. For H2 atmosphere incubation the cabinets were evacuated four times to 20 lb vacuum, introducing H2 each time to 3 lb. This resulted in an H2 atmosphere containing about 0.5 per cent 02. Palladium-impregnated asbestos was used as a catal- yst to reduce remaining traces of 02. Small tubes of methyl- ene blue agar, visible through the glass door, were used to indicate the establishment of reducing conditions. Partial or complete replacement of H2 with CO2 was effected only after reducing conditions had been established. Aerobic plates were incubated on the laboratory table covered with a sterilized cloth. Both aerobic and anaerobic incubations were carried out at 28 to 30C. After various incubation periods, growth was estimated visually on a scale of 0 to 4. Cross-streak inoculations were made and appropriate notations for syner- gism or antagonism were used. Black and white Polaroid photographs proved to be useful as a permanent visual record of growth and inter- action responses. Attempts to record pigmentation contrasts on various types of color film were not successful. 34 Bartholomew and Mittwer's "Cold" method spore staining procedure (21) was used to identify spores. Microbial Counts From Simazine Treated Plots An experiment was designed to investigate effects of simazine on soil microbial populations. Soil samples were taken in the fall of 1966 from an orchard pesticide experi- ment on Hillsdale fine sandy loam.4 Duplicate samples were taken of untreated soil and soil which had received 4 1b sima— zine each year in 1964, 1965, and 1966. In 1966, 2 lb. amitrole—T was combined with the simazine. Each sample was composited from three field randomized plots, four core samples in each plot. Samples were sifted through a 6 mm sieve, placed in plastic bags and stored in a cold room at 40C for about a week before estimates of microbial numbers could be made. An estimate of the total population of fungi, actinomycetes, and bacteria in untreated and treated soil was obtained by use of soil dilution plate counts. The actinomy- cete population was estimated after 10 days growth on chitin agar (53), the fungal population after 7 days on Martin's Rose Bengal agar (61), and aerobic bacteria after 8 days on soil extract agar (55). Anaerobic bacteria were estimated 4Courtesy Dr. S. K. Ries, Department of Horticulture, M.S.U. The experiment was located on the University Farm, East Lansing. 35 on media 103, 105, and 106 (Table 2) incubated in 100% H2 or 90% C02- 10% H2 for 14 days. Bacteria suppressed by the H2 and C0 but still viable, were estimated by counting the 23 additional colonies which had appeared 3 days after removal from anaerobic conditions. All incubations were at 28 to 30C. Denitrification In Liquid Broth In an effort to establish nitrate-reducing and de- nitrifying capacities of the eighteen pure culture bacteria (Table 1) in the presence and absence of simazine, a study was undertaken using liquid media in Durham fermentation tubes. Media 103, 105, 106, and 113 were employed (Table 2). In preparing media 103, 105, and 106 the ingredients were combined to form two separate solutions. The first solution containing buffer solutions, mineral salts, and nitrate or asparagine was sterilized by autoclaving at 121C for 15 minutes. One-half of each medium contained bromthymol blue indicator. Five m1 of this first solution was dispensed in— to Durham tubes (outer tube, 16 x 125 mm: inner vial, 10 x 75 mm, inserted upside down) by means of a sterile automatic syringe. The second solution, containing glucose, vitamins, amino acids, and iron chelate, was sterilized through a millipore filter into a suction flask maintained at 45C in a water bath. Sufficient simazine, dissolved in chloroform, was added to the appropriate quantity of each medium through 36 the Millipore filter to achieve the desired final medium concentration. Vacuum at 45C was employed to remove the chloroform before this second solution was added to the Durham tubes, using a sterile automatic syringe. Medium 113 (Timonin's medium, 95) was prepared and dispensed similarly, with appropriate variations. All tubes were put into anaerobic chambers and re- ducing conditions Obtained by evacuating and filling the chambers with H2. During the process the gas trapped in the inner vial was evacuated and the vial filled with the re- spective medium. Each bacterial culture was prepared for inoculation by growing for 18 hours in nitrate broth. To minimize carry- over of nutrients, 0.10 ml of each turbid culture was pipetted aseptically into 10 ml of a sterile solution formed by combining solution's A and B (Table 2) in a ratio of 7:3. The resulting pH was 7.3. This suspension was mixed and 0.10 ml inoculated into each Durham tube. The tubes were placed in anaerobic incubators. To disperse the inoculum through the medium, and also to provide anaerobic conditions, the anaero- bic chambers were evacuated and filled with N2. The cultures were allowed to incubate for 10 days at 28 C in this N2 atmosphere. For each organism, duplicate tubes were incubated at each factorial combination of four media with and without bromthymol blue, with and without simazine (5 ppm). 37 During incubation, the tubes with the indicator, ini- tially light blue because of the inclusion of bromthymol blue, changed to an intense blue as the solution became more alkaline, an indication of denitrification, or to a yellow or green as the solution became more acid. This reaction change was recorded as "alkaline" if the color was intense blue, or "acid" if yellow or green, or "—" if unchanged. A second indication of denitrification was the pro- duction of gas which was collected in the inner vial. Gas production was estimated visually on a scale from - to +++. A third observation was the test for microbial re- duction of nitrate to nitrite. Approximately 1.0 ml of sul- fanilic acid solution and a 1.0 ml of dimethyl—alpha- naphthylamine was added to each of the tubes which did not contain bromthymol blue. The development of a red or maroon color indicated the presence of nitrite. A negative test (no development of color) could be interpreted to mean, either that nitrate was not reduced, or that nitrate reduction had occurred but the reaction had gone beyond the nitrite stage to ammonia or gaseous nitrogen. Therefore, it was necessary to test for nitrate in those tubes giving a nega- tive reaction for nitrite. This was done by the addition of a very small amount of powdered zinc. Zinc reduces nitrate to nitrite, and the color characteristic of a positive nitrite test developed if the nitrate had not been completely reduced. RE SULT S Cultural Studies on Solid Media Pure culture studies were undertaken with eighteen species and strains of nitrate—reducing bacteria. Obser- vations of their behavior on different media and under vari- ous atmospheric conditions were made in an effort to es- tablish a differential counting procedure, using solid media. The test organisms selected represented different physiologi— cal types based on studies by WOldendorp (111) and Valera and Alexander (100). The chemically defined media of WOldendorp were used with slight modifications (see Table 2). Buffer concentration and the concentration of Ca and Mg were re- duced to avoid precipitation of phosphate and iron. The vitamin component was augmented by addition of inositol and vitamin B12 as in media described by Lochhead and Chase (56). Plates were streaked by loop inoculation with washed cell suspensions from log phase cultures grown in nutrient broth. A major difficulty in preliminary anaerobic studies was the complete elimination of traces of oxygen at the be— ginning of incubation. Traces of oxygen can permit initi— ation of growth and adaptation to anaerobic conditions in 38 39 the case of some facultative anaerobes (Valera and Alexander, 100). To prevent this the plates were chemically reduced by storage in H2 prior to streaking and precautions were taken to minimize the length of time that media were exposed to air during inoculation. Responses to inositol and thioglycollate The major difference in media used in this study and the comparable media used by woldendorp (111) was the addi- tion of vitamin B12 and inositol to the list of vitamins sup— plied as growth factors. A preliminary study was designed to investigate effects of inositol on growth of representative test organisms when added at the rate of 50 mg per liter as in media described by Lochhead and Chase (56). The possi- bility was also investigated that sodium thioglycollate might contribute to clearer differentiation of anaerobic responses by minimizing oxidation during the time that plates were necessarily exposed to air while being streaked. Aerobic growth responses of four bacterial strains are presented in Table 3. All organisms made better growth with asparagine as source of nitrogen (medium 110) than ni- trate (medium 109). With nitrate as nitrogen source, three of the four organisms made more rapid growth when inositol was left out of the complement of growth factors (medium 114). The further elimination of thioglycollate in medium 115 pro- duced no further changes in growth. 4O .wumHHoqumofleu ssHoOm no Houamocs “soapss moH I mHH soaowz .HopamocH usoouas ooH I eHH assume .AmumHHooszoHeu.EsHoom + Hop IHmOCH mchsHoeH mcHEmpH> + mpHUm OCHEm + ochmHmmmm + mmoosHmv I OHH Eswpwz .AmumHHoohHmOHnu EBHUOm + HouHmocH mCHpsHocH mcHEmuH> + mpHom OGHEm + opmnuHe + wmoosHmV I NOH EBHUQZQ .m on 0 mo mHmom m :0 NHHmsmH> pmumfiflumw £p3onwm m m m m a x m o memmH ease I. mchHmauuHcmo .2 .6H m H m H a m H o oomeH come I. wasnomacmsoHH .m .oH m m m m a m m o AonH mooH.I mCMHsmmoo .m .n m N m N m m m N QHOUGOUHOZII. mmocwmduom .mm .N mmmo v amp H mmmp e awn H mwmp e amp H mmmp e map H mHH EsHpmz eHH EsHowz OHH EsHomz 00H EsHpmz EchmmHO .OmN um QmHUmE “mom :0 mausonm UHQOHQN .m mHnma 41 Observations of growth under three different anaerobic atmospheres are presented in Table 4. Of the four organisms, only_g§. aeruginosa was unable to grow in the absence of nitrate (medium 110) in a 100 per cent H atmosphere. A11 2 made very good growth on this medium in atmospheres contain- ing high partial pressures of CO although growth of.M. 2) denitrificans was depressed when 10 per cent H2 was included to assure catalytic reduction (on palladium) of traces of oxygen in the C02. In the three media supplying N as nitrate, the inosi- tol in medium 109 strongly suppressed growth of M. denitrifi- cans in the 100 per cent H atmosphere, and to some extent 2 growth of the two Bacillus species also. This effect was not expressed in the two CO atmospheres. Elimination of thio— 2 glycollate in medium 115 promoted a release of growth of M. denitrificans in 100 per cent CO and some reduction in 2 growth of_g§. aeruginosa in this atmosphere. During the course of this experiment, it was observed that reducing conditions could be established in methylene blue agar within one hour after replacement of air with H2 in the presence of the palladium catalyst. The time required to streak a group of plates for incubation was no more than one and one-half hours. Thus, the total exposure of experi- mental inocula to aerobic conditions was something less than two and one-half hours. There appeared to be no advantage to use of thioglycollate, so it was eliminated from media used in later studies. 42 Table 4. Anaerobic growtha on agar mediab at 28C. =====================z— =======z Medium Medium Medium Medium Organism 109 110 114 115 100% H2C 2. EE' aeruginosa Woldendorp 2 O 2 2 7. g. coagulans 2 2 3 3 l4._§. lichiniformis ATCC 14580 2 2 3 3 l6._M. denitrificans ATCC 13543 % 1 3 3 100% cozC 2._g§. aeruginosa WOldendorp 2 3 2 l 7. g. coagulans 1963 II(W) 3 4 3 3 14. B. lichiniformis ATCC 14580 3 4 3 3 16. M. denitrificans ATCC 13543 3 4 2 4 90% co2 — 10% H26 2. Es. aeruginosa WOldendorp l 3 l 1 7. g. coagulans 1963 II(W) 3 4 3 3 14. g. lichiniformis ATCC 14580 3 4 3 3 16'.M~ denitrificans ATCC 13543 3 2 3 3 aGrowth estimated visually on a scale of 0 to 4. bMedium 109 - (glucose + nitrate + amino acids + vitamins including inositol + sodium thioglycollate). Medium 110 — (glucose + asparagine + amino acids + vitamins including inositol + sodium thioglycollate). Medium 114 — 109 without inositol. Medium 115 - 109 without inositol or sodium thioglycollate. c15 days growth. 611 days growth. 43 In spite of the evidence in Tables 3 and 4 that inositol influenced growth behavior of some species un— favorably under certain atmospheric conditions, it was re— tained in later media as being a growth factor likely to exist in soils. Later experience suggests that the concen- tration may have been so high as to be unnecessarily re- strictive, particularly for estimation of aerobic elements in soil populations. Interactions of nutrition, atmospheric composition and simazine onggrowth To study growth, pigmentation and organism inter- actions, the selected test organisms were streaked on agar5 media 103, 105, and 106 (see Table 2) and incubated in aero- bic, H or CO2 atmospheres. Following the anaerobic incu— 2: bations, plates from H2 and CO2 were incubated under aerobic conditions to allow recovery of the test organisms. Because_g§. aeruginosa [organism 1] had been ob- served in earlier studies to obtain good growth under ad- verse nutritional conditions and also had shown strong antagonism to several other organisms, it was streaked vertically on the plates as a standard test organism. Three organisms were streaked at right angles to_§§. aeruginosa on each plate to allow each to express possible interactions with this organism. 5DIFCO Bacto Agar was used. 44 Growth responses for 18 organisms tested are tabu- lated in Table 5. Growth of Achromobacter hartlebii [organ- ism 15] was erratic on all test media. Unique cultural re- quirements of this organism were not satisfactorily resolved. Under aerobic incubation, most organisms grew best on medium 106 with vitamins, amino acids, and asparagine as the nitrogen source (Table 5). Aerobic growth of all organisms was restricted on glucose-nitrate without growth factors (medium 103). _§§. aeruginosa [organisms l and 2],_M. denitrificans [l6] and_g§. fluorescens [17] made distinctly better aerobic growth than the others on this medium but did respond to the growth fac- tors in medium 105. Marked responses to growth factors were expressed by §. marcescens [4], several Bacillus strains [5, 6,8,9,10,l4] and by.§§. denitrificans [l8]. _§§. stutzeri [3] and the slower growing_§. macerans [11,12] and g. circulans [13] were less responsive to growth factors when confined to nitrate as source of nitrogen. However, growth patterns and requirements changed when grown under anaerobic atmospheric conditions. Without oxygen, other compounds or elements must act as electron ac- ceptors to enable growth. With the organisms studied, ni- trate effectively promoted growth under strongly reductive anaerobic conditions. This can.be observed in Table 5 where medium 105 with nitrate, vitamins, and amino acids proVided the best medium for growth in an H2 atmosphere. 45 Table 5. Growtha of denitrifying bacteria on solid mediab in various atmospheres. ___— m Aerobic H2 H2 + CO aerobic — a _ OrganismClOB 105 106 103 105 106 103 105 106 103 105 106 1 3 4 5 2 2 l 2 3 5 2 3 5 2 3 4 5 2 2 k 2 2 5 2 4 5 3 l 2 4 1 2 0 1 2 1 0 % 0 4 l 3 5 2 3 1 2 3 3 1 2 3 5 l 3 3 2 3 1 2 3 1 l 2 4 6 l 4 5 3 4 2 3 4 4 l 4 4 7 2 3 4 4 5 2 4 6 3 2 4 5 8 l 4 5 2 4 2 2 4 4 l 4 4 9 1 4 5 2 3 l 2 4 3 1 3 5 10 l 4 5 2 5 2 2 6 5 % 2 5 ll % 1 2 2 3 l 2 3 1 2 2 3 12 % l 2 2 3 3 3 3 3 2 2 3 l3 % 1 3 2 3 2 3 4 2 1 2 3 14 1 4 5 3 4 2 3 4 4 1 4 4 15 -— -- -- -- -- -- —- -— -- —- -- -- 16 3 4 5 2 3 1 2 3 3 0 5 l 17 3 4 5 0 0 0 0 0 2 % % 0 18 1 4 5 1 2 1 l 2 2 2 3 5 6 aGrowth at 28C estimated visually on a scale of 0 to bMedium 103 - (glucose—nitrate). Medium 105 - (glucose + nitrate + amino acids + vitamins including inositol). Medium 106 - (glucose + asparagine + amino acids 1 vitamins including inositol). CSee Table la dan HAA4+4nn=1 nrnw‘I'I'I 'Ih (10'. ul- aerobic. 46 Medium 106 with asparagine, vitamins, and amino acids produced growth with most organisms in H2, though less than in air, and with organisms 8,10,12,13, and 18 growth was equal to that in the basic nitrate medium without growth factors. With the probable exception of organisms l,2,3,l6 and 18, all of the strains tested are known to have ready ac— cess to non-respiratory (fermentative) pathways of metabolism. However, it must be recognized, also, that the sparse growth observed visually and designated in the table by values of l or % may have occurred at the expense of electron acceptors which accumulated, exogenously or endogenously, by chemical or enzymatic oxidations during the 1% hours that the plates were exposed to air while being inoculated. The fact that respiratory pathways are used preferentially for growth by at least half of these organisms is demonstrated by their rapid growth on medium 106 when plates were removed from H2 into air for three days. .g. coagulans [organism 7] produced moderate to heavy growth on both nitrate media under H2. The three g. licheni- formis strains [6,8,14] and one of the B. cereus strains [10] also grew well using nitrate in the presence of growth factors. These and all other organisms listed, except the first three and the last three, were able to utilize nitrate more effectively in H2 than in air when growth factors were not supplied. With H incubation, gs. fluorescens [l7] grew only 2 on the asparagine medium and then only after exposure to air. 47 This indicates that the H2 killed the inoculum on nitrate media and residually inhibited or delayed growth with aspara- gine. A number of other organisms may have been similarly inhibited residually by H2 on one or both nitrate media, since they failed to make additional growth during the subse- quent three day exposure to aerobic conditions. By contrast, only organisms 5,11,12, and 13 failed to make additional growth on the asparagine medium when exposed to air. These were all bacilli (B. laterosporus, g. macerans and-g. circulans). With CO2 incubation, all organisms were either resi— dually inhibited by exposure to the 100 per cent CO2 atmos- phere, or growth made in CO2 exhausted the medium of some com- ponent essential for growth. At any rate, no additional growth occurred during subsequent aerobic incubation, so only observations made at the end of the incubation in CO2 are recorded in Table 5. During incubation in CO2 most organisms produced their greatest growth with asparagine as the nitrogen source. The CO atmosphere greatly inhibited organisms 3,16, and 17 2 on all media. For a number of others, CO2 was a less favor- able atmosphere than H2 for growth on either nitrate medium. For most organisms, CO2 was more favorable than H2 for growth when asparagine was the nitrogen source. Except for organisms 3,16, and 17, growth in CO2 was amazingly similar to growth under aerobic conditions on all three media. 48 Stotzky and Goos (91) found that more than 90% CO2 reduced the number of organisms developing on soil extract dilution plates, but most organisms recovered when the plates were subsequently incubated in air. From this observation and the present study it may be inferred that, if any of these test organisms (except numbers 3,16, and 17) were present in the soil utilized by Stotzky and Goos, they would have fully develOped on their media while in CO2 incubation. The differential responses to nitrogen source and growth factors observed here under different atmospheres provide ad— ditional criteria for isolation and enumeration of more re— stricted physiological groups within the soil population. Simazine effects on the growth of the pure culture test organisms were noted after 2 and 12 days' incubation under H2 and 90% H2 + 10% C02 atmospheric conditions on solid media 107,109, and 110. Simazine was supplied at 0 and 50 ppm in the media. After 2 days' growth under H2 incubation several organisms responded with a slight increase in growth on both nitrate and asparagine media treated with simazine. However, after 12 days' incubation only a few Bacillus species still indicated a better growth response on simazine media. With the inclusion of 10% C02 in the H2 atmosphere, this growth response to simazine was limited to a very few Bacillus species on the nitrate media after 2 days' growth and was diminished after 12 days' incubation. Thus it appears that 49 simazine may have decreased the lag phase for some of the test organisms, especially under 100% H2 atmospheric con- ditions, with a very slight increase in total growth on the nitrate media. This apparent tendency for simazine to promote more rapid development on solid media is consistent with the re- duced lag times and more rapid growth observed with_§§. aeruginosa,_§§. stutzeri and §. marcescens by Hong in broth cultures at 30C when amino acids were left out of a medium containing glucose, nitrate, vitamins and minerals (see Figure 3). It should be noted that this adaptive stimulus was reversed at lower temperatures and that simazine was strongly inhibitory at 10C (cf Figure 4). Interactions of nutrition, atmospheric composition and simazine on antagonism by Pseudomonas aeruginosa The physical and chemical characteristics of soil, both natural and man—induced, determine the nature of the en— vironment in which microorganisms are found. These varying environmental characteristics in turn affect the composition of the microbial population both quantitatively and quali- tatively by producing numerous ecological niches. These niches are essentially situations of opportunity for exploi- tation by individual species whose evolutionary and physio- logical adaptations give them a unique advantage in that par- ticular environment. 50 In this study only a few of the stimuli to which com- ponents of the soil microflora respond will be considered, including nutritional factors, micrObial interactions, atmos- pheric conditions and the effect of the herbicide, simazine. In natural soil environments, a number of relation— ships exist between individual microbial species. Members of the microflora may rely upon others for certain growth substances, but at the same time they may exert detrimental influences on still other components. Beneficial, harmful and neutral associations can be demonstrated in isolated systems. Their significance in complex natural soil systems is much more difficult to evaluate. Nevertheless, the numerous studies that have been made support the general eco- logical principle that the composition of the microflora of any soil habitat is governed by the biological equilibria created by the associations and interactions of all indi— viduals found in the population. It is well realized that the results of.ig vitro studies with pure bacterial cultures on chemically defined media cannot be directly assumed to apply to natural situ- ations. However, some indication of possible interactions between bacterial species may be observed and general tenden- cies inferred for well defined nutritional and atmospheric conditions. Although man is an animal, the environmental stimuli which derive from his technological inputs are both quanti- tatively and qualitatively different than those of any other 51 animal. His use of pesticides is a unique example of quali- tatively new and different stimuli being introduced into natural environments. The pesticide technologies in vogue are based upon their most obvious effects on target species. The potential significance to man of their effects on non- target species and on systems of species important to him has been a serious concern to researchers for a much longer period of time than it has to the general public. With reference to the soil microbial population, ef- fects of pesticides on taxonomic groups have frequently been negligible when tested at 10 to 100 fold normal rates of application. It appears likely that specific effects on species or physiological groups of bacteria, fungi or actino- mycetes may be masked by compensating changes in other groups or species. If such specific effects are expressed, it will be necessary to look for them in these more restricted groups. At some point in such research, it will be necessary to look for effects on individual species and interactions between them. The pure culture studies reported here were under- taken to look for differential effects of simazine on repre— sentative denitrifying species. Different nutritional and atmospheric conditions were imposed with a view to defining parameters for differentiating physiological types within the group for later population and isolation studies in field experiments. 52 In preliminary studies by Sheng (85), it was ob— served that several nitrate reducing bacteria in the col- lection exerted antagonistic effects on others. Both gs. aeruginosa strains were particularly active in this regard, but the ATCC strain was active against a larger number of other strains, including the WOldendorp strain of_g§. aerugin- osa. It appeared useful to set up pure culture studies in such a way that effects of simazine on individual species and on this interaction with_g§. aeruginosa ATCC 10145 could be observed at the same time. Accordingly, organisms were cross—streaked against _g§. aeruginosa [organism 1] so that interactions with this standard organism might be observed. As can be seen in Table 6 growth inhibition of several organisms by organism 1 occurred under aerobic incubation. The complete nitrate medium 105 provided conditions most suitable for a wide spectrum of growth inhibition, since only_§. cereus [10], _gg. fluorescens [l7], and_g§. denitrificans [18] failed to be inhibited. However, the most extensive inhibition oc- curred with the three.§. licheniformis strains [6,8, and 14] and_§. coagulans [7], as shown in Figures 5 and 6, on the basal nitrate medium 103. Other instances of growth inhibition occurred under CO incubation with_§§. aeruginosa [2] on all test media 2 (see Figure 7), and B. coagulans [7] on media 103 and 106 (see Figure 8). 52—A I HHJUH IOS MEDIUM I06 Fig. 5. Aerobic growth and interactions of_§§. aeruginosa .(strain 1) and_§. licheniformis (strains 6, 8, and 14). MEDIUM I03 I HEJUH IOS AEROB I C 28C MEDILM IOG Fig. 6. Aerobic growth and interactions of_g§- aeruginosa (strain 1) and_§. laterosporus [5],_§. coagulans [7] and_§. cereus [9]. 52-B HHNUHIOO Fig. 7. Growth and interactions in C02 of_g§. aeruginosa (strains 1 and 2),_§§. stutzeri [3] and_g§. fluorescens [l7]. IEDUHIOS MEDIUM |06 Fig. 8. Growth and interactions in C02 of_§§. aeruginosa (strain 1) and B. laterosporus [5], g. coagulans [7] and_§. cereus [9]. 53 In contrast to the strong inhibition of g. licheni- formis [6,8, and 14] under aerobic incubation, the ATCC strain of gs. aeruginosa [l] markedly stimulated growth of these B. licheniformis strains under H2 on media 103 and 105 (see Figure 9). Similar stimulation to growth of several organisms was observed during growth in H2 and in C02. In most cases, this commensalistic effect disappeared or, at least, was seldom enhanced and never reversed during subse— quent growth in air (see Table 6). In C02, the stimulation was most pronounced on Medium 105. A comparison of Figures 9 and 10 illustrates the marked release of growth which occurred with many organisms on medium 106 (no nitrate) when removed from H2 into air (see Table 5). Test organisms were also cross-streaked against_g§. aeruginosa [l] on solid media with O, 5, 25, and 50 ppm sima- zine. Variations occurred in the intensity of antagonism by _g§. aeruginosa against the test organisms at the different simazine levels. On the nitrate media under aerobic conditions, the antagonistic action of organism l against_g§. stutzeri [3], _§. marcescens [4], g. laterosporus [5], and B. licheniformis [8] was increased when simazine was applied, but its antagon- istic effect against B. coagulans [7] under these same con- ditions was decreased. Under H2 conditions on nitrate media, the antagonistic effect against g. laterosporus [5] increased 53-A MEDIUM I03 W Y” HYDROGEN W . 28 C -———-—v- MEDIUH IOG Fig. 9. Growth and interactions of_g§. aeruginosa (strain 1) and B. licheniformis (strains 6, 8, and 14) in a hydrogen atmosphere. MEDIUM I03 I \ k ’H \ HYDROGEN 8 DAYS (I ; AEROBIC 2 DAYS. I w. \ ....n‘ MEDIUM I06 Fig. 10. Growth and interactions of_g§. aeruginosa (strain 1) and_§. licheniformis (strains 6, 8, and 14) after 2 days in air following 8 days in hydrogen. 54 .AHODHmocH mCHpsHUGH MCHEmuH> + mpHom osHEm + DGHmmHmmmm + mmousHmw I 00H EsHpmz .HHouHmocH msHpsHUcH mcHEmuH> + mpHom OGHEm + mpmuuHc + omoosHm .HmpmupHc + wmoosHmv I MOH EsHpmz I moH ssHomz Q .meHOH OOH< mo £u3oum mo mpHEHH Eoum Demume EE 0m Op m pm>u0mno mwmcomm Ion NHODMHsEHum no 0>Hmmmummsm meHucommumwn .e on H mo mHmom 0 so NHHmsmH> pomeHummm H H N H H N H H N H N N N H N H N m N N H H N H H H m m m N H N H N N e N H N H H N N H N N H N H m N w H N v H m N v H N N N m N N H mH NH 0H 0H NH 00H mOH mOH 00H mOH MOH oOH mOH NOH oOH mOH MOH oOH mOH mOH oOH mOH NOH oOH mOH mOH EMHcmmHo COHumHseHum cOHumHBEHum Echommu:< COHumHBEHum EMHcommu:¢ COHumHseHum QEMHcommusm UHQoumm + N00 N00 UHQOHDM + Nm Nm DHQDHmm .moumHm Hmmm pomeMDMImmouo so monogamoEum msOHHm> nope: HmeHOH Ooafiv mmosHmsuom mmGOEOpsmmm an mHkuomQ mCHNHHHuHcmp msOHHm> mo 6 coaanssHum cam coHDHnHecH enzouo .m OHQMB 55 in the presence of simazine, but decreased against_§. macer- _§§§ [11 and 12]. In general the influence of simazine on microbial interactions was not distinct and requires more investigation. Interactions of nutrition and atmospheric composition on pig- ment production byj£§ggggmgg§§ aeruginosa _g§. aeruginosa is well known for its production of several pigments, as well as colorless compounds, with anti- biotic properties. Several investigators have shown that pyocyanin, a blue pigment, inhibits certain organisms, es- pecially gram-positive bacteria. Others have defined nu— tritional requirements for pyocyanin production. These inr clude magnesium, alanine or several other amino acids, and Krebs cycle intermediates. In the antagonism studies described in the previous section, pigment production by_g§. aeruginosa was found to be dependent upon the nitrogen source, and the amount of pig- ment was influenced by the atmosphere of incubation. 'With nitrate as the nitrogen source, as in medium 103 without vita- mins and amino acids and in medium 105 with vitamins and amino acids, a blue-green pigment was produced by one strain of_§§. aeruginosa only (ATCC 10145), and then only during aerobic incubation following H2. No pigment was produced during or after exposure to CO2 on either nitrate medium. Under com- pletely aerobic conditions, the blue pigment was produced only on medium 105. However, with asparagine as nitrogen 56 source in medium 106 with vitamins and amino acids, both ATCC 10145 and the WOldendorp strain of §§° aeruginosa [organ- isms l and 2 respectively] produced a yellow-green pigment in air. The intensity of the pigment increased in the following order: aerobic < aerobic succeeding C02 < aerobic succeed- ing H2. It was observed that even under the most favorable conditions for production of yellow—green pigment by ATCC 10145, the pigment did not appear when this organism was growing near several of the other test organisms. To observe this phenomenon, ATCC 10145 was cross- streaked against each of the remaining test organisms on medium 106 and incubated for 8 days in H2, followed by 4 days in air. As a control,_g§. denitrificans [18L which allowed intense development of yellow-green pigment by ATCC 10145, was streaked against this Pg. aeruginosa strain on each plate together with one other organism. As noted in Table 7, g. marcescens [4] and several bacillus organisms, including_§. laterospgrus [5],_§-.ggggg- ‘lgng [7], B. cereus [9 and 10], g. macerans [11 and 12], and .g. circulans [l3] prevented visible formation of pigment. Growth of ATCC 10145 was not affected, but the typi- cal yellow-green pigment did not appear in a diffusion zone extending 5 to 20 mm beyond the limit of growth of the organ- isms producing the effect. No attempt was made to determine whether production of the pigment was inhibited or whether 57 Table 7. Inhibition by various denitrifying bacteria of pig- mentation in Pseudomonas aeruginosa (ATCC 10145) on solid mediaa after exposure to an H2 atmosphere. Organism Pigment inhibition 2. _g§. aeruginosa Woldendorp no 3. _g§. stutzeri ” " 4. S. marcescens MSU-MPH yes . laterosporus 468 B(W) " licheniformis 430 (W) no . coagulans 1963 II(W) yes . licheniformis Pl (W) no . cereus ATCC 6464 yes 10. . cereus ATCC 14579 " 12. . macerans ATCC 8244 " 13. . circulans ATCC 4513 " l4. . licheniformis ATCC 14580 no 15. . hartlebii ATCC 365 " 16. .E .3 .1} g g g 11. .B. macerans ATCC 843 " g £5 .5 A _M. denitrificans ATCC 13543 " P 17. s. fluorescens ATCC 11250 " 18. _g§. denitrificans ATCC 13867 " aMedium 106 — (glucose + asparagine + growth factors - see Table 2). bPigment inhibition after growth for 8 days in H2 plus 4 days in air. 58 the pigment was actually produced but not in its chromatic form because of potentiometric or ionic effects. The remaining test organisms had no observable ef- fect on pigment production by the ATCC strain of_g§. aeruginosa. There was no apparent relation between pigmentation phenomena and the antagonistic or stimulatory responses as- sociated with proximity to ATCC 10145. Thus, the most ex- tensive inhibition occurred on media 103 in aerobic con- ditions where_g§. aeruginosa [1] failed to produce a visible pigment. Also organisms 6, 12, and 13, which were inhibited under aerobic conditions where pigments were produced by gs. aeruginosa ATCC 10145, were stimulated by this organism under H2 and subsequent aerobic incubation where pigments were also produced. Ps. aeruginosa strains are known to pro- duce several antimicrobial products, some of which are pig- ments, some which are not [24]. It is entirely probable that different antibiotics with different spectra of activity, are produced under different nutritional or environmental conditions. On the other hand, pyocyanin has been implicated in electron transfer [24,33,35]. It is possible that exogenr ous redox systems involving pyocyanin may be poised favorably for a given organism under anaerobic conditions but unfavor- ably under aerobic conditions. These results serve to illustrate the complexity of interactions which may occur in natural soil habitats where 59 an extremely heterogenous mixed inoculum is always present to respond to environmental and nutritional stimuli imposed by weather or the activities of plants, animals or man. Population Studies with Soils Treated in the Field with Simazine Plus Amitrole-T Stotzky and Goos (90) demonstrated that segments of soil microbial populations could be characterized on solid media by their ability to tolerate high CO tensions and/or 2 low 02 tensions. High CO2 tensions were more restrictive than low concentrations of 02. Tolerance to high CO2 was most prevalent among fungi and least characteristic of actinomy- cete species in the microflora of soils which had not been conditioned previously in atmospheres high in CO2 and low in 0 With such prior conditioning, however, the increase in 2. numbers of COz-tolerant bacteria and actinomycetes was much greater than the increase in tolerant fungi. It was not es- tablished whether these increases were due to enrichment of specific groups which were already tolerant or whether physiological adaptation had occurred. The denitrifying microflora in soils are, charac— teristically, facultative anaerobes. For some denitrifying species, the adaptation to anaerobic conditions involves a shift to non-respiratory (fermentative) pathways of metabolism for which nitrate is not essential, although nitrate may be reduced coincidentally if it is present. For others nitrate is essential, in the absence of oxygen, as a terminal 6O electron acceptor in respiration. It is generally agreed that the latter type of physiological adaptation is charac- teristic of the most active denitrifying species (Delwiche, 25; Alexander, 1; woldendorp, 111; Verhoeven, 101). The term, denitrification, refers specifically to the dissimilatory reduction of nitrate to gaseous products (principally N2 and N20) which are lost from the soil. Adaptive mechanisms which lead to these losses include the synthesis of the non-constitutive enzyme, nitrate reductase. Studies of Ries and Tweedy (77) have shown that simazine stimulates nitrate reductase activity in higher plants. Were this effect to be expressed on a major component in the microflora of the soil or rhizosphere, it could alter patterns of nitrogen transformation and transport external to the plant root in ways which might materially influence the nutrition of the plant. The pure culture studies of Hong, in broth media, which were cited in the Literature Review, and the studies on solid media described in earlier sections of this report are consistent in supporting the View that simazine does, in fact, promote the adaptation to utilization of nitrate by a number of bacterial species that have been implicated in de- nitrification losses of nitrogen from soils. There is reason to suspect that exposure of a soil population to sima- zine will, over time, produce changes consistent with an en- richment in nitrate reducing capacity. 61 A major objective of the present study was to in- vestigate cultural parameters which might be employed to de- tect such population changes by dilution plating on solid media, rather than by the tedious, statistically equivocal, most probable numbers technique which involves serial di- lution in broth media. Accordingly, the orchard experiment described earlier (p. 34) was selected as a source of soil samples. Dilution platings were made on solid media to estimate numbers of the major taxonomic groups (bacteria, actinomycetes and fungi) and numbers of bacteria which might respond uniquely to nu- tritional and atmospheric variables employed in cultural studies described in previous sections. Dilution platings were made of each duplicate field sample which had been taken to represent treated and un— treated soil-—a total of four soil samples. For each soil sample and each medium, five plates were poured for each of four dilutions. Colonies were counted on 3 to 5 plates of the significant dilution (55). Colony counts, factored for dilution, were converted to logarithms for analysis of variance. Table 8 shows that the probabilities for no difference between mean logarithms for control and treated soils ranged from 5 to greater than 50 per cent. This generally low level of significance for treatment differences was due to unexplained field variation, rather than to excessive variability in counting and plating procedures in the laboratory. Standard deviations for 62 Table 8. Microbial population estimates and statistical probabilities for differences between controls and soils with previous history of field treatment with simazine plus amitrole-T. Mean log numbers per g dry soil . Simazine Incubation . a atmosphere Medium Control _p1us S.D. t P amitrole—T Air 118 5.4371 5.3651 0.123 0.585 50 " 117 6.1211 6.0025 0.090 1.318 50 " 116 7.7030 7.2265 0.023 20.717 5 " 111 7.0835 6.4159 0.139 4.803 20 C02 b 103 5.7240 5.5749 0.055 2.711 30 " plus air " '6.5287 6.3749 0.144 1.349 50 C02 105 5.7649 5.6310 0.138 0.970 >50 " plus air " 6.2817 6.2188 0.148 0.425 >50 C02 106 5.7536 5.4524 0.085 3.544 20 ” plus air " 6.2199 5.5465 0.420 1.603 40 H2 103 5.6913 5.4610 0.131 1.758 40 " plus air " 5.6645 5.2794 0.071 5.424 20 H2 105 5.7222 5.5847 1.009 0.136 >50 " plus air " 5.5259 5.1499 0.014 26.857 5 H2 106 5.5166 5.4834 0.028 1.186 50 ” plus air " 5.7076 5.6185 0.238 0.374 >50 aMedium 116, soil extract agar (55); medium 117, chitin agar (53); medium 118, rose bengal agar (61); media 103, 105 and 106--see Table 2. b H2. Plates exposed 3 days in air after 8 days in CO Only new colonies which developed in air were count 5d. or 63 dilution plate counts within soil samples ranged between 2 and 5 percent of the mean, whereas standard deviations for field variation not associated with replication or treatment ranged up to 19 per cent of the mean. For future studies, it appears essential to increase field replication in sampling. In spite of the low order of statistical significance for most comparisons, certain relationships between the different combinations of media and incubation conditions should be considered seriously for further study. Geometric mean numbers are presented in Table 9. From the last column of this table, it can be seen that the general effect of chemical treatment was to reduce numbers in the major taxonomic groups and in most cultural sub-groups capable of growth on media 103, 105 and 106. Fungi were re- duced 15 per cent, actinomycetes by 25 per cent, and bacteria on soil extract by 66 per cent. This reduction in bacteria was significant at the 5 per cent level of probability. The completely synthetic medium 111 was much more re- strictive for bacteria under aerobic conditions than the soil extract medium 116. However, 111 is the appropriate complete medium for comparing effects of nitrogen source and growth factors in 103, 105 and 106 (see Table 2). Total bacteria capable of growth on medium 111 were almost 80 per cent less in treated than in control soil. The distribution of cultural sub-groups within this bacterial population was drasticly different in treated and untreated soil. This can'be seen more clearly in Figure 11. PER CENT OF TOTAL AEROBIC COUNT ON MEDIUM Ill Fig. IOOP 90- 80 70" 60" 50~ 40 3O 20 IO 11. T I T I I 63-A IN IN 645 All? CONTROL |'_'] TREATED s //////////////// IIII!Illllllll|I||I||III|I|I||||||IIIIIIIIIIIIIIIIIIIIIIIIII|IllllllIIIIIIIIIIIIIIIIIIIIIIIlIllllllllllllllIIIIIIIIIIIIIIIllllllIlllIIlllI|llIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIllIIIIIIIHIII: ’////////////A |I|IIlllllllllllllllllllllllllllI|II|III|I|l||IllIlIlllIIllIllllIlllIIIl||I|I||I||I|II|||IIIIIIIIIllllIlIllI|IIIllIIIIIIIIIIIIIIIIlllllIIII|II|I||IIlIIlIllllIIIIIIlllIllIlIIIIIIIII|IIIIl|IIIIIlIIIlIIIIIIIIIllIIlIllIllIllIl|llIIIIlllIlIlllllllllllllllllllllIlllIIIIIIIIIII 7////////////A IIIIIIIIIIIIII 7////////A Illlllllllllllllllllllllllllllllllllll W |||||||||||||||||||I|I|||||I||||||||||||||||II 7////////// |I|||I||||l||||||IllI \ 00z IIz co H, co IIz MEDIUM |03 MEDIUM l05 MEDIUM I06 Effects of field treatment with simazine plus amitrole—T on the distribution of cultural groups in the bacterial population defined by aerobic plate counts on medium 111. 64 Table 9. Geometric mean numbers and relative numbers of microbial groups in control soils and soils with previous history of field treatment with simazine plus amitrole—T. m Geometric mean numbers per g dry_soil Treated Simazine soil as Incubation a Microbial plus %.of atmosphere Medium group Control amitrole-T control x104 x104 Air 118 Fungi 2.7 2.3 84.8 " 117 Actinomy— 13.2 10.0 76.1 cetes " 116 Bacteria 504.7 168.5 33.4 " 111 Bacteria 121.2 26.1 21.6 CO2 b 103 " 5.3 3.8 71.0 " plus air ” " 33.8 23.7 70.4 C0 105 ” 5.8 4.3 73.4 " plus air I. II 1901 1606 8605 CO 106 " 5.7 2.8 49.9 " plus air " " 16.6 3.5 21.3 H2 103 " 4.9 2.9 58.8 " plus air " " 4.6 1.9 41.3 H2 105 ” 5.3 3.8 72.9 " plus air " " 3.6 1.4 42.1 H2 106 " 3.3 3.0 92.6 " plus air " " 5.1 4.2 81.5 aMedium 116, soil extract agar (55); medium 117, chi- tin agar (53); medium 118, rose bengal agar (61); media 103, 105, and 106--see Table 2. bPlates exposed 3 days in air after 8 days in CO2 or H2. Only new colonies which developed in air were counted. 65 In Figure 11, the assumption is made that organisms which developed on Media 103,105 and 106 would have also de- veloped aerobically on medium 111 which contained the growth factors and both nitrogen sources which were systematically eliminated to achieve the more restrictive nutritional con- ditions represented by the other three media. This assumption is open to criticism, but it provides the basis for a useful first approximation. In the control soil, only 20 to 35 per cent of the bacterial population defined by aerobic growth on medium 111 was able to grow and/or survive in CO2 when nitrate was the only source of nitrogen (in media 103 and 105). In treated soil, it appeared that this population consisted almost en- tirely of COz-tolerant types. When asparagine was substituted for nitrate as a nitrogen source (medium 106), differences in CO2 tolerance between control and treated soils were neglibi— ble, considering the weak basis for statistical inference. However, it should be pointed out that the proportion of bacteria capable of growth in CO2 (bottom bar segments in Figure 11) was consistently greater in treated soils on all three media. It is recognized that a 100 per cent CO2 atmosphere is an unrealistic environment. It is possible that the com- mercial source of C02 used may have carried ppm traces of oxygen (this was not checked). Nevertheless, enhanced 002 concentrations are certainly present in the vicinity of de- composing biological detritus (plant, animal or microbial) and in the rhizosphere. As Stotzky and Goos (90) have 66 pointed out, tolerance to CO is a demonstrable ecological 2 factor which has received inadequate attention in soil micro- biology. The relationships between CO2 tolerance and treat— ment of soils with simazine and/or amitrole-T which appear in Figure 11 need to be taken seriously in terms of future research. A very pertinent consideration in this regard is the fact that the major increase in CO2 tolerant bacteria in treated soil appeared to involve aerobic types which could only use nitrate for rapid growth after removal from CO2 into air. It may be postulated that these were organisms in which dissimilatory nitrate reduction may have supported a mainten- ance level of respiration to permit them to survive but not to grow and multiply in the absence of air. When air was supplied, rapid assimilatory nitrate reduction, supporting rapid growth, was possible because of an enhanced nitrate reductase activity which appeared to be rather clearly re- lated to chemical treatment. Unfortunately, two chemicals were involved, simazine and amitrole-T. Further studies to investigate this relationship with both chemicals are needed, using experimental designs which will provide a more reliable basis for statistical inference. A 100 per cent H2 atmosphere is less realistic than one containing only C02. It was used in this study to assure a strongly reducing environment. It is of interest that the proportion of the total population which was able to grow in 67 this atmosphere on all three media was greater in treated than in control soils, as it was with 002. The ability to grow in one or both atmospheres was shown by a number of the denitrifying species and strains that were used in the pure culture studies described in earlier sections of this report. Similar studies need to be carried through to characteri— zation of isolates to determine the extent to which nutrition- al and atmospheric variables can be combined to permit esti- mation of denitrifying populations by dilution plating techniques. An incidental observation of significance for future investigations was the appearance on a number of plates of medium 103 of a fungus which was isolated and identified as a Specie or strain of Trichoderma viride. The fungus was found only on plates inoculated from the soils treated with simazine and amitrole-T. It was found capable of vigorous growth on a mineral medium with simazine as sole source of carbon and nitrogen. Later studies on medium 103 showed that, while spore germination, growth and sporulation oc- curred rapidly in air, only spore germination and limited growth occurred in a C02 atmosphere. In an H2 atmosphere, germination of spores and growth of mycelium were inhibited. On removal from C02, a ring of spores formed quickly along the periphery of growth made previously in C02, and sporulating mycelium developed rapidly to cover the remainder of the plate. After removal from H2, spore germination occurred 68 promptly and growth and sporulation proceeded rapidly to cover the plate. Under some conditions, not clearly de- fined, a concentric pattern of alternating white vegetative hyphae and green sporulating mycelium would appear. Effects of Nutritional Parameters and Simazine on Growth and Denitrifi- cation in Broth Media The growth studies of Hong in broth cultures (cited in the Literature ReviewIindicated that simazine acted both to reduce the lag period for adaptation to nitrate and to in- crease the rate of growth after adaptation. Observations on development of streak cultures on solid media were consistent with this interpretation. Inocula used by Hong were grown in a broth medium in which amino acids were the sole source of both carbon and nitrogen. Inocula for the studies on solid media were grown on nutrient broth (peptone plus beef extract) amended with yeast extract. In both groups of studies, simazine effects were observed in test media in which glucose (or citrate in mediumljgn'were supplied as carbon sources. The observed ef- fects of simazine on lag periods and rates of growth could have been due to effects on adaptive enzyme changes involved in glucose metabolism as well as the inferred stimulus to de— velopment of nitrate reductase activity (woldendorp, 111). These relationships and their significance for actual losses O of nitrogen by denitrification need more critical study. 69 As a first step in this direction, an experiment was designed to determine whether simazine effected growth or de— nitrification in the presence of glucose by organisms in which nitrate reductase activity had been induced by prior cultivation in the absence of glucose but in the presence of nitrate. Accordingly, cultures were adapted by serial trans— fer in nitrate broth (Difco: peptone, beef extract, KNOz). Eighteen-hour cultures in this growth medium were used to inoculate test media dispensed in Durham fermentation tubes. Test media included 103, 105, 106, and a standard medium for denitrifiers (medium 113, after Timonin, 95). Bromthymol blue was added to a separate series of all media to observe changes in reaction (Valera and Alexander, 100). Simazine, at the rate of 5 ppm, was added to a second com— plete series of all media, with and without bromthymol blue. Duplicate tubes of each composition were inoculated and incubated at 28C in an atmosphere consisting of 90 per cent N2 plus 10 per cent H2. Observations made after 10 days are recorded in Tables 10 to 13. With reference to the main objective of this experi- ment, the most significant observation is that 5 ppm simazine had no effect on response of any of these adapted cultures. Several incidental observations of value for future work should be pointed out. A serious error in procedure was the failure to dis- tinguish in the growth notation in column 2 between positive 70 .H 6369 ammo .OsHumeHm Emu m mo mocmmnm Mo wosmmmum O30 CH Damn mzu OM03 pm>n0mno momeommwmn H.N OHQmB 00mg .MHmecHE + ODOHDHG + omoosHOm OCHHOMHG +++ +++ I + + NH I I I +++ + + NH OGHHOMHN +++ +++ I + + OH I I I I I + mH UHUN I I +++ + + eH pHud I I I I + MH 304 I I +++ + + NH 304 I I +++ + + HH pHoa I I +++ + + OH pHUfi I I +++ + + m pHod I I +++ + + m pHod I I +++ + + n pHod I I +++ + + o UHom I I +++ + + m OGHmeHfl I I +++ + + e OcHmeHe +++ +++ I + + m OGHHOMHN +++ +++ I + + N OGHHOMHG +++ +++ I + + H COHpomwu coHuospoum GOHuospoum COHuosponm COHuospwm MM.mmcm£O One I. new ODHMUHZ oumHqu nuSouO Dawn HoE>£quum SUHB osHm HOE>£quum usonuHS mmwmflmmmw U H.0cHNmEHm usoauHB pew £0H3 meuoun mOH EsHpOE GH OHHOUUOQ mcHhmHuuchp mo noncommmn HmuspHsu .OH OHQOB 71 . H 0369 mmmo .OCHNCEHm Ema m mo OUCOQO Ho OUCOmem OCH CH mEmm may OHOB Um>Hmon mmeommwm Q AN. OHQOB momv .MHOHOCHE + muouomm Cu3oum + mumuuHC + OmoosHOm OCHmeHm +++ +++ + + + NH mCHmeHfl I I +++ + + NH OCHmeHm +++ +++ + + + 0H I I I I I + mH pHom I I +++ + + 6H I I I I I + mH oHon I + +++ + + NH CHUC I I +++ + + HH I I + +++ + + OH I I + +++ + + m pHo< I I +++ + + m UHo¢ I + +++ + + o I I I +++ + + o I I I +++ + + m OCHmeHC + + +++ + + e OCHmeHC +++ +++ + + + m OCHmeHC +++ +++ + + + N OCHmeHm +++ +++ + + + H CoHuomOH COHuosoonm COHuoscoum COHuospoum COHuospOm CH.OmCmao mmw mmw OUHMUHZ OUCHUHZ Cusonw wsHm HoENCquum CDHB wsHm HoENCuEOHm DCOCHHB Ommwmmmmm. "HIIIHIII I I III III ili i' Illl H.0CHNmEHm UCOCDHB pCm CuH3 mnuonn mOH EsHpoE CH OHHDUUOQ mCHmeHuHCOC mo momCOQmmH HmHCuHCU .HH OHQOB 72 .OCHHmMHm mfimoon HopmH ECHCOE .NH EMHCmmHo CHHSC . H OHQCB mom .OCHNmEHm Emu m mo OOCOQO Ho OUCOMOHQ OCH CH wEmm OCH wuw3 pw>HOon mmmCommOm A.N OHQmp womv .mHmHOCHE + mnouomm Cuzonm + OCHmmummmm + OmoosHO U Q m I I I I I + NH I I I I I + 5H I I I I I + 0H I I I I I I mH I I I I I + CH I I I I I + NH ppHum + +++ I I + NH pHu< I ++ I I + HH I I I I I + OH I I I I I + m I I I I I + w I I I I I + h I I I I I + m I I I I I + m CHUC + + I I + e I I I I I + m I I I I I + N I I I I l + .... COHuomOH COHuospoum COHuospoum COHposponm CoHuospOm CH OmCmCO mmw mmw OHHHUHZ wumuqu Cu3ouw OCHm HOE>CpEonm CDHB wsHm HOEHCHEOHm psOCpHZ oflMHfiMdmm. H E A.mCHNmEHm HCOCHHB pCm COH3 mCuoun oOH ECHCOE CH OHMODUOQ mCHNHHHUHCop mo momCommon HOHCHHCU .NH OHQOB 73 . H OHQOB OOmU .OCHNOEHm Emm m Ho OUCOOHO Ho OUCOmOHm OCH CH OEmm OCH OHO3 UO>HOmHo OOmCommOmH .mHOHOCHE + OCHmOHmmmm + OHOHHHC + OHOHHHU "A may EBHOOE CHCOEHBm OCHHOHHC +++ +++ I + + NH I I I I I + NH OCHHOHHC ++ I I + + OH I I I I I I mH I I I +++ + + «H I I I I I I NH I I I I I I NH I I I I I I HH I I I +++ ... + 3 I I I +++ + + o I I I +++ + + m I I I +++ + + H I I I +++ + + o I I I ++ + + m I I I +++ + + e OCHHOMHC +++ +++ I + + m OCHHOHHC +++ +++ I + + N OCHmeHG + + I + + H COHHUOOH COHHospoum COHHUCUOHm COHHUCOOHN CoHHospOm MM.OmCOCO mmO MOO OHHHHHZ OHOHHHz CH3OHO OsHm HOE>CHEOHm CHHz OsHm HOEHCHEOHm HCOCHHK ammmmdmm. U H.OCHNOEHm HCOCHHS pCm CHH3 OCHOHH MHH ECHUOE CH mHHOHomH mCHmHHHHHCOC Ho OOmCommOu HOHCHHCU .mH OHHOB 74 growth and questionable growth evidenced by only slight turbidity (Tables 10 to 13). A positive growth notation should be questioned in these tables if there is no other evidence of activity in the culture. Thus, it is unlikely that g. circulans [13] developed significantly in any medium except 103, where its activity was evident because of the in- crease in acidity apparent with bromthymol blue. _A. hartlebii [15] was as problematic in its growth in these broth cultures as it was on solid media. _g§. aeruginosa [1 and 2] and_§§. denitrificans [18] behaved clearly as reported by others. They were unable to grow (slight turbidity) in the absence of nitrate on medium 106. In the other three media, they grew vigorously, re- duced all available nitrate and produced gas and a strongly alkaline reaction with bromthymol blue. The production of both gas and alkalinity are necessary as evidence of de- nitrification (100). _M. denitrificans [16] grew and denitrified vigorously on glucose, both in the absence of amino acids and vitamins (medium 103) and in their presence (medium 105). It behaved in the same way for woldendorp in vaspar—sealed tubes (lll). Valera and Alexander (100), however, found this organism un- able to grow on glucose in a purified N2 atmosphere unless amino acids were also supplied. Its ability to do so in the present study can be accounted for by the presence of 10 per cent H2 in the N atmosphere. .N° denitrificans is faculative- 2 1y capable of chemolithotrophic growth, utilizing H2 as an 75 energy source in the presence of nitrate (48). The extent to which the 10 per cent H2 in the incu— bation atmosphere may have influenced the behavior of other organisms in the test is a matter for further investigation. A number of responses were inconsistent with those reported by other researchers. For example,_g§. stutzeri [3] has been found incapa- ble of using glucose or a number of other carbohydrates un- less amino acids are also supplied. In the present study, organism 3 grew and denitrified as vigorously on medium 103 as did.g§. aeruginosa [1 and 2] or.£§. denitrificans [18]. Hong (see p. 12) had observed that this strain behaved differ- ently than the ATCC strain of_g§. stutzeri with which he be- gan his investigations. Its identity is obviously question- able, although in numerous cultural test it has complied with other criteria for Pg. stutzeri. The possibility that H enrichment of the atmosphere may influence behavior of 2 this organism needs to be investigated. A number of Bacillus species are reported incapable of anaerobic growth on glucose and nitrate in the absence of amino acids or growth factors. Here, on medium 103, they were found capable of sparse growth, reducing nitrate to ni- trite in the process and producing acids. There was some evidence of gas production by several of these strains when amino acids and vitamins were supplied in medium 105, but none produced alkalinity as confirming evidence for denitrifi- cation on any medium. woldendorp had reported gas production 76 as evidence of denitrification for several of these bacilli when amino acids or amino acids plus vitamins were supplied with glucose and nitrate (lll). Valera and Alexander did not find the confirming increase in alkalinity in similar media for_§. licheniformis, the only Bacillus species in their tests. NOne of these investigators checked specifically for reduction of nitrate or production of nitrite. _§. marcescens [4] was not studied by any of these re- searchers. It is of interest that it gave evidence of de- nitrification on medium 105 (production of gas and alkalinity) and presumptive evidence of denitrification on medium 103 (production of alkalinity without gas). On citrate without growth factors (medium 113) it produced neither gas or alka- linity. A similar observation may be made in the case of_g§. fluorescens [17], which produced alkalinity (but no gas) only on medium 105. Wbldendorp (111) has proposed that glucose may be a more discriminating energy source for differentiating be- tween true denitrifiers (such as gs. aeruginosa) and those which produce N2 or N20 coincidentally to fermentation (such as the Bacillus species) than are glycolytic intermediates and by—products or Krebs cycle intermediates. His suggestion is based on his own observation and those of others that B. licheniformis is unable to denitrify on glucose as energy source, whereas Verhoeven (101) had classified it as a true denitrifier on the basis of its behavior in a medium supplying 77 glycerol as energy source. All of the Bacillus species in woldendorp's tests were able to use glycerol for growth and produced gas anaerobically. Because of its availability to a large proportion of the soil microflora, glycerol is probably the energy source of choice for estimating total numbers of denitrifying organ- isms, as is indicated by studies of Valera and Alexander (100). The usefulness of glucose for discriminating between true and coincidental denitrifiers within the total group of denitrifiers is strikingly illustrated by the results which are presented for media 103 and 105 in Tables 10 and 11. Equally clear definition on solid media will be more diffi- cult to obtain because the criterion of gas production will be less readily observed. Production of alkalinity appears to provide a basis for differentiation which could be ex— ploited on solid media. woldendorp found that the oxidative deamination of glutamic acid was closely linked with nitrate reductase activity in_g§. aeruginosa but not B. licheniformis. The possibility needs to be investigated that glutamic acid may be a more specific and discriminatory energy source than glucose for differentiating between true and coincidental denitrifiers. DI SCUSSION The present investigations were undertaken to define nutritional and atmospheric parameters which would permit use of dilution plate counts on solid media to detect changes in nitrate reductase activity in soil populations and to differ- entiate between important types of nitrate reducing organisms. The population changes observed in simazine treated field soils suggest that dissimilatory nitrate reducers can be distinguished from organisms which only reduce nitrate assimilatively for growth on the basis of the ability of the former to grow in a COZ-enriched atmosphere on solid media supplying nitrate and the ability of the latter to grow on the same media only after removal from CO2 into air. On the other hand, the parameters tested do not pro- vide any basis for differentiating on solid media between organisms, such as_g§. aeruginosa, in which anaerobic growth involves respiratory pathways dependent upon nitrate for terminal electron transfer, and the Bacillus types which de- rive their energy under anaerobic conditions from fermentation reactions which may or may not be accompanied by reduction of nitrate to gaseous products. In broth culture, the two criteria of gas production and increased alkalinity do clear- ly differentiate between these "true” and ”coincidental" 78 79 denitrifiers when glucose is used as the energy source. How- ever, even in broth culture, a given organism may fail to denitrify on glucose but do so when some intermediate or by- product of glycolysis, such as glycerol, is present in the medium. The nature of growth factors supplied can further complicate the picture. It is apparent that no specific energy source has yet been identified for making the distinction between these two types of dissimilatory nitrate reduction, in either broth or solid media, in a way that clearly relates to soil con- ditions and the types of substrates which may be expected to predominate in specific ecological situations such as the rhizosphere. Wbldendorp (111) suggests that glutamic acid may be uniquely involved as a hydrogen donor in reduction of nitrate to gaseous products. This amino acid is found in root exudates. Its possible usefulness for making a meaning- ful distinction between types of nitrate reducers should be investigated, both in broth and solid media. A serious difficulty with agar media is the fact that strictly defined nutritional conditions cannot be achieved because of impurities in even highly purified agar. In the present study, growth ratings of % to l on streak cultures may have been due in part to metabolites present as impurities in the agar. These impurities become an even more serious factor when highly restrictive agar media are used for count— ing purposes. Due ’ allowance should be made for the contri- bution of the agar to colony counts in interpreting numerical 80 estimates on solid media. There are distinct advantages in the use of solid media for the purposes entertained in this study. Atmos— pheric composition in the immediate environment of organisms developing in thin layers of agar can be more precisely con- trolled than in tubes of broth. The possibility of observing the effect of changing the atmosphere cannot be entertained in broth culture. The release of growth after removing cultures from CO2 into air in the present study provided the unique criterion for distinguishing nitrate reduction from those which reduce nitrate assimilatively for growth. With careful standardization of suspending and dilution pro- cedures, the statistical reliability of plate counts is much greater than of the most probable numbers treatment of ob— servations obtained from serial dilution. The relationships reported here between field treat- ment and the distribution of cultural sub-types in the bac- terial population need to be verified by further studies. For one thing, the fact that amitrole—T was included with the simazine treatment in the third year makes it impossible to ascribe the observed effects specifically to either compound. More field replication is needed, also to give more acceptably low probabilities for error. However, there was clear evidence that simazine stimulated more rapid adaptation to use of nitrate in pure cultures of bacteria. The increase in the proportion of nitrate utilizing bacteria in field populations exposed to 81 simazine is consistent with this_ig vitro behavior. The fact that the major proportionate increase was in types that appeared to be not active denitrifiers suggest that simazine may act in the field to conserve nitrate nitrogen by promoting its temporary immobilization in microbial tissues. If this action can be verified, it may provide another useful appli- cation for this chemical. SUMMARY Cultural parameters were investigated which might be useful in detecting changes in nitrate reductase activity in soil bacterial populations. A.major concern was to develop solid media which could be used for isolation and enumeration of nitrate reducing bacteria by dilution plating rather than by serial dilution in broth. Equally important was the search for criteria which could be used to differentiate be— tween three types of organisms: those in which nitrate re- duction is (1) essentially assimilatory and those in which it is (2) essentially or (3) coincidentally dissimilatory. Three parameters were considered: (1) nitrogen sources, (2) growth factors, and (3) incubation atmospheres. Nitrate and asparagine were compared as nitrogen sources. Media with no growth factors were compared with media pro- viding both vitamins and amino acids. Aerobic responses were compared with anaerobic responses in C02, H2 or N2, alone and in various combinations. Glucose was used as the carbon source, except as comparison was made with a standard medium for denitrifiers in which the energy source was citrate. Simazine, a herbicide known to stimulate nitrate re- ductase activity in higher plants, was tested for its ability to promote the same adaptive response in 18 strains of known 82 . E I . . -.Iul-...MUI,II . AU HQI ... Emma.- ICE... 83 faculative nitrate reducing bacteria. The observed responses of these pure strains to simazine in the various cultural situations which were imposed were used as the basis for inferences regarding population estimates obtained under parallel cultural conditions for field soils previously treated with simazine. Several distinct relationships were observed which support the validity of the objectives undertaken and point to useful directions for further research: 1. Simazine introduced into solid media at concentrations of 5 to 50 ppm promoted more rapid development of streak cultures of several nitrate utilizing bacteria. These obser- vations supported earlier studies, under this project, in broth culture. 2. This stimulus to growth appeared to be due to earlier adaptive synthesis of nitrate reductase, since there were no effects on growth or denitrification when simazine was added To broth cultures previously adapted to nitrate. 3. Previous annual applications of simazine over a three- year period in the field reduced plate counts (relative to un- treated controls) for major taxonomic groups in the order: bacteria (66%) > actinomycetes (25%) > fungi (15%). 4. The field simazine treatments increased, strikingly, the proportion of nitrate reducers in the bacterial population. This was evidenced by increased numbers of colonies capable of growth and/or survival in 100 per cent CO2 or H2 atmos- pheres on media supplying nitrate. 84 5. The major proportionate increase in nitrate reducing bacteria, however, occurred among types which differed from representative pure strains of denitrifying bacteria in their ability to grow on nitrate media only after removal from CO2 into air. It is inferred that these are types in which nitrate reductase activity, stimulated by simazine, is exploited for assimilatory reduction of nitrate to support growth. 6. Field treatment with simazine produced negligible proportionate changes in bacterial types capable of growth and/or survival in CO2 or H2 on a medium which did not cone tain nitrate. 7. It appeared that a clear distinction could be made between dissimilatory and assimilatory nitrate reducers on solid media supplying nitrate on the basis of the ability of the former to grow in CO2 and the ability of the latter to grow only after removal from CO2 into air. The cultural parameters employed with solid media did not provide criteria for distinguishing between true denitrifiers and coincidental dissimilatory nitrate reducers as clearly as is possible in broth media. 8. Specific areas are identified for further research to define cultural parameters more specificly in relation to unique physiological processes. The need for greater replication in field studies to provide a broader base for statistical inference is indicated. fiuflmflnfll : E. .HH-BIN.» A I... I. I. ...” ... 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