. -——q-«.—-—.o Mvw— WA _- _ NITROGEN. ‘FIXATION BY AZOTOBACTER IN A NATURAL AQUATIC ENVIRONMENT Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY SARAH JOSEPHINE CHAPMAN 197 2 I ' fl . . ~ . I . '6‘14“ 5‘.) 4' 11/14.. Aux LIBRARY UniVetsity =1’ algae? Y :3 ' ”MAGS-$53 .BUUK BINDER‘T NC. “I LIBRARY BINDERS ............. rum“ ,_—_—~ ABSTRACT NITROGEN FIXATION BY AZOTUBACTER IN A NATURAL AQUATIC ENVIRONMENT By Sarah J. Chapman. Azotobacter is a nitrogen fixing bacterium. Laboratory studies imply that Azotobacter plays a significant role in the nitrogen cycle and consequently it appeared important to study the relative importance of this genus in nitrogen fixation in the natural environment. Wintergreen Lake at the W. K. Kellogg Biological Research Station was the lake site used for in Situ experiments. Nitrogen fixation was assayed using the acetylene reduction technique. Viable counts were made. Radioisotopes were used to study respiration and protein synthesis. The glucose level in the lake was estimated using the HobbieAWright kinetic analysis of the uptake of radioactive glucose by the natural lake pOpulation. Most probable number studies were made to determine the numbers of Azotobacter in the lake. Azotobacter vinelandii appeared not to contribute significantly to nitrogen fixation in Wintergreen Lake water. Studies of viability, respiration, and protein synthesis in lake water indicated that cells added to lake water retained essentially a maintenance level metabolism. Suitable carbon sources appeared to be limiting in the lake, with the glucose concentration only 13.5 ug/liter. Mbst probable number studies Sarah J. Chapman showed that aerobic, heterotroPhic nitrogen fixers were present in very low numbers in the lake, about one bacterium in two milliliters of lake water 0 NITROGEN FIXATION BY AZOTUBACTER IN A NATURAL AQUATIC ENVIRONMENT By Sarah Josephine Chapman A THESIS Submitted to Michigan State University in partial fulfillment of the reQuirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1972 FT\‘ ACKNOWLEDGEMENTS The author wishes to thank Dr. J. M. Tiedje for his encourage- ment and patient guidance throughout the period of this work. Special thanks are due to Dr. T. R. Corner, Dr. M. J. Klug, and M. Louise Brock for their constructive criticisms of the manuscript. Special thanks are also due V. K. Hitchens for her help on experiments on nitrogen fixation during encystment and respiration and protein synthesis by Azotobacter in lake water. Words cannot express the thanks due the many friends who endured chilly winds, frostbite, and cold, damp, dark nights on the lake to lessen the hours spent boating and eavesdropping on the geese. The author is especially thankful to her parents for their patience, support, and encouragement. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . . . . 5 Lake Site . . . . . . . . . . . . . . . . . . . . . . . . . 5 Assay for Nitrogen Fixation . . . . . . . . . . . . . . . . 5 (l) Gases. . . . . . . . . ._. . . . . . . . . .‘. . 5 (2) General procedure. . . . . .'. . . . . . . . . . 5 (3) Analysis . . . . . .-. . . . . . . .'. . . .-. . 6 Bacterial Cultures. . . . . ._. . . . . . . . . .-. . . . . 6 Correlation of Ultrastructure with Nitrogen Fixation. . . . 6 Isolation and Classification of Azotobacter from Wintergreen Lake. . . .'. . . .‘. . . . . . . . . . . . . . 7 Growth of the Two Isolates of Azotobacter chroococcum from Wintergreen Lake Under Various Partial Pressures Of oxygen 0 0 O 0 O O O O O O O O O O O I O 0 O O O O O O O 8 Preparation of Cells Used in Experiments on Nitrogen Fixation in Lake Water, Viability in Lake Water, Repres- sion of Nitrogen Fixation by Ammonium Ion, and Nitrogen Fixation During Encystment. . . . . . . . . . . . . . . . . 9 Preparation of Bacteria-Free Lake Water . . . . . . . . . . 9 Nitrogen Fixation in Lake Water . . . . .p. . . .d. .'. . . 9 Viability in Lake Water . . . . . . . .'. . . . . . . . . . lO RadiOisotopeS O O 0 O O O O O O O 0 O O O O O O O O O O 0 0 10 Respiration Studies . . . . . . . . . . .-. . . . . . . . . 11 (1) calls 0 O O I O O O O O O O O O O O O O O O O O O 11 (2) supernatant o o o o o o o '0 o o o 0 O O o ‘0 o o o 11 (3) C020 0 a 0‘0 0 o 0'. o o o o o o o o 0‘. o o e o 11- Protein synthESiS o o o o I. o o o o o o o o o o o I. o _o o o 12 Amount of Glucose in Lake Water . . . . . . . . . . . . . . 12 iii Page Repression of Nitrogen Fixation by Ammonium Ion . . . . . . 13 Nitrogen Fixation During Growth . . . . . . . . . . . . . . 13 Nitrogen Fixation During Encystment . . . . ._. . . . . . . 14 Most Probable Numbers (MPN) of Azotobacter in Wintergreen Lake 0 a o o o I. o o o o o o o o o o o o o o o 0 14 RESULTS. 0 o o o o o o o o o o o o o o o .' o ‘0 o o o '0 o o o o o u 15 DISCUSSION 0 o o o o o o o o o o o o o o o o o o o o o o o o o o o 42 LITERATURE CITED 0 o o o o o o o o o o '0 o o o 'c o o o o o o o o o 55 APPENDIX 0 o o o o o o o o o o o '0 o o o .. o o o o o o o ‘0 o o a a 59 iv Table The effect on acetylene reduction of flushing with an gas mixture to remove N2. Ar, 02 N2 fixation by Azotobacter vinelandii in lake water: , and CO 2 laboratory studies. LIST OF TABLES O O 6 Viability of Azotobacter vinelandii in lake water . . Respiration studies of Azotobacter vineZandii in lake water 0 o o O Uptake of radioactive glucose by a natural lake population. N2 fixation by Azotobacter vinelandii and Azotobacter chroococcum in medium with and without NH Cl. 4 Most probable numbers of Azotobacter in Wintergreen Lake. Utilization of various carbon sources by Azotobacter. . . Acetylene reduction during the growth of 2 strains of Azotobacter chroococcum under various partial pressures of oxygen . O O Page 15 17 18 21 23 35 35 36 LIST OF FIGURES Figure Page 1 Ethylene production by Azotobacter'vinelandii vs time . . . l6 2 Respiration of cells in lake water and in Burk's buffer . . l9 3 Protein synthesis in lake water vs buffer . . . . . . . . .- 22‘ 4 Graphical analysis of bacterial uptake at low substrate concentrations following MichaeliséMenten enzyme kinetics. Plot of Cut/c against increasing substrate concentrations (8), illustrating derivation of 1) maximum natural substrate concentrations (Kt + Sn) as ug/l; 2) maximum velocity of bacterial uptake (V) as pg per liter per hour; and 3) turn- over time for substrate regeneration (Tt) as hours. . . . . 24 5 Ultrastructure of Azotobacter vinelandii grown with N2 as the nitrogen source. Total magnification is 87,000X. Bar represents 0.21.1111 o 0 do 0 o o o o ‘0 o o o o o o o e o o 25 6 Ultrastructure of Azotobacter vinelandii grown With NH Cl as the nitrogen source. Total magnification is 10 ,OOOX. Bar represents 0.2 pm. ... . . . . . . . . . . . 26 7 Ultrastructure of.Azot0bacter vinelandii grown with N2 as the nitrogen source. Total magnification is 34,500X. Bar represents 005 pm 0 o o o ‘0 o o o .o 0 .° 6 .0 O '6 o ‘o o o 27 8 Ultrastructure.of.Azot0bacter chnoococcum grown with NH4C1 as the nitrogen source. Total magnification is 26,000X. Bar represents 1 pm . .‘. . . . -.- .‘. . . .'. .1 28 9 Repression of ethylene production in Azotobacter vinelandii by ammonia nitrogen. . ._. . . . . . . °.° . . . 29 10 The effect of NH4+ on ethylene production by.Azot0bacter vinezmdii With time 0 O l. O O O O 0. O O I O C I O O O O O O O 30 ll Ethylene production during growth of‘Azotobacter vinelandii in Burk's buffer plus 1% glucose . . . . . . . . 32 12 The effect of fresh medium on ethylene produced . . . . . . 33 13 N fixation during encystment . .‘. . . .‘. . . .'. . . . . 34 2 14 fixation during growth of.Azot0bacter chroococcum sgrain l isolated from an ethanol enrichment. . . . . . . . 37 vi Figure 15 l6 17 18 19 20 N2 fixation during growth of Azotobacter chroococcum strain 2 isolated from a glucose enrichment . .'. . . Growth of Azotobacter chroococcum isolated on glucose under various partial pressures of oxygen . . . . . . Growth of Azotobacter chroococcum isolated on ethanol under various partial pressures of oxygen.. . . . . .' Gas flow apparatus used in studies of growth under various partial pressures of oxygen . . . . . . . .‘. A sterile sampling device for lake water. . . . . . . Plexiglass holder for incubating samples in the lake. vii Page 38 39 40 -41_ 6O 61 INTRODUCTION Nitrogen is an essential element. Its transformations in the biosphere are almost completely regulated by aquatic and terrestrial microorganisms. The availability of nitrogen compounds can.govern the degree of photoautotrophic develOpment in numerous waters (1). The ability of an organism to fix atmospheric nitrogen, i.e., to convert N2 to 2NH3, may serve as a selective adVantage in nitrogen-poor environments. A nitrogen fixing organism, Azotobacter, was isolated by Beijerinck and van Delden in 1902 (3). Later workers have shown that pure cul- tures of Azctobacter actively fix nitrogen in amounts as large as 20 mg per gram sugar (15,46), which is more than double that fixed by most clostridia (1). Although laboratory studies imply that Azotobacter plays a significant role in the nitrogen cycle in the environment, definitive‘in situ experiments have not been performed. Azotobacters are free-living, aerobic, heterotrophic microorganisms widely distributed in soil and water (27,34). They are characterized morphologically by the generally large size of the individual cells which vary considerably in shape and size (34,48). Some azotobacter species have the ability to form cysts, modified vegetative cells with thick coats, under conditions not favorable for vegetative cells (29, 30,34). Considerable information is available concerning the morphology, growth requirements, carbon metabolism, and utilization of nitrogen 2 sources by Azotobacter (7,8,9,lO,1l,l3,15,l6,17,l8,l9,21,22,26,34,35, 39,41,45,48,51). The specific influence of a combined nitrogen source on nitrogen fixation varies with the quantity and quality of combined nitrogen present. Azotobacter will both fix nitrogen and utilize ammonia nitrogen when the latter is present in low concentrations, i.e., 150 mg ammonium per liter (8,39,42). Azotobacter will also fix nitrogen. when low levels of nitrate are present, provided molybdenum and vanadium are present in quantities sufficient for nitrogenase synthesis; in the absence of these metals, only nitrate is used (5,21). Higher concentra- tions, i.e., 410 mg/liter, of ammonium inhibit nitrogen fixation by Azotobacter (8,11,26,39). The order of preference of utilization of nitrogen sources is: (l) ammonia, (2) urea, (3) nitrite, (4) nitrate, and (5) atmospheric nitrogen (10,39). Therefore, one would expect Azotobacter to be fixing nitrogen in significant amounts only when a source of combined nitrogen is unavailable. Organic nitrogen compounds, except urea, are utilized less readily than ammonium, nitrate, and free nitrogen (19). Azotase is the enzyme complex in Azotobacter which catalyzes the fixation of molecular nitrogen, the amount of nitrogen fixed being directly proportional to the metabolic activity of the cell (7). Azotobacter requires phosphorous, sulfur, potassium, calcium, magnesium, iron, and molybdenum, in addition to some form of nitrogen.and an energy source, for growth. If one of these substances is present at suboptimal concentrations or lacking, the organism.will have a reduced growth rate or exhibit no growth, consequently showing a decreased rate of nitrogen fixation or complete absence of nitrogen fixation (4,7,18, 22,23,26,38). Cobalt has been shown to stimulate the amount of nitrogen 3 fixed (24). Azotobacter can utilize a wide variety of carbon sources (13,15,17,26,4l). Nutritional requirements alone do not determine the ability of Azotobacter to grow and fix nitrogen.- Other factors such as temperature, oxygen concentration, pH, and interaction with other organisms combine with nutrient levels to form a dynamic system in which conditions can be favorable or unfavorable for Azotobacter growth and nitrogen fixa— tion. Azotobacters are mesophilic with minimum, Optimum, and maximum temperatures for growth of approximately 10, 30, and 40-45 C (26). The temperature optimum may vary depending on the nitrogen source (7). The nitrogen~fixing efficiency of Azotobacter increases with a decrease in p02 (36,42). The optimal pH for Azotobacter growth is around pH 7.2- 7.6 (51.). The minimal pH at which growth can still be maintained varies from pH 5.5-6.5, depending on the strain (39). Nitrogenase, the nitrogen fixing component of the azotase complex, functions best over a fairly narrow pH range near pH 7.0, and fixation falls off markedly above and below about pH 6.5-7.0 (9). It was reported that nitrogen- fixation by Azotobacter was stimulated by the presence of associated organisms (26), but Lind and Wilson (31) concluded that media or the techniques or both were unsatisfactory for the best deve10pment of the organism. The role of associated microorganisms may be only to alter the medium so that it becomes more suitable for the growth of the Azotobacter. Since heterotroPhic nitrogen fixation depends on the availability of exogenous organic carbon, the presence of other organ- isms may also have a negative effect on growth and nitrogen fixation by Azotobacter. In the natural environment, Azotobacter may not be able to successfully compete for available carbon sources with other nitrogen-fixing organisms, and also with non-nitrogen—fixing forms. 4 In the present study, the ability of Azotobacter vinelandii (ATCC no. 12837) to fix nitrogen in Wintergreen Lake water was investi- gated in an attempt to determine the importance of Azotobacter vine- Zandii in the nitrogen cycle of the lake. The studies established that Azotobacter vinelandii does not.fix nitrogen in Wintergreen Lake water and therefore probably does not contribute significantly to the nitrogen cycle. MATERIALS AND METHODS Lake Site Wintergreen Lake is a eutrophic lake at the W. K. Kellogg Bio- logical Research Station, Hickory Corners, Michigan. Assay for Nitrogen Fixation Nitrogen fixation was assayed indirectly by a modified acetylene reduction technique of Stewart, Fitzgerald, and Burris (43). (1) Cases. Azetylene (purified grade), a gas mixture of 02 (22%), C02 (0.04%), and argon (78%, high purity), and certified ethylene standards of 47 ppm and 830 ppm ethylene in nitrogen were obtained from the Matheson Co. (2) General procedure. Experiments were carried out in 6.0 ml glass serum bottles fitted with rubber serum stoppers. Two milliliters of the sample were added to each bottle. Air was removed by flushing the liquid with the premixed Ar, 02, C02 gas phase for about one minute, and.each bottle stoppered. After injection of 0.5 ml of acetylene by means of a glass syringe, the samples were incubated for one hour at 25 C in the laboratory or in the lake. Reactions were terminated by the injection of 0.3 ml of 2.0%-HgCl2 (w/v). Samples were assayed in duplicate, along with one zero-time control. The atmosphere in the headspace.was analyzed for ethylene produced. The amount of ethylene produced resulted from a one hour incubation time; 6 however, it was recorded as the amount produced at the initial time of sampling. (3) Analysis. Ethylene concentration was determined with a Varian-Aerograph Model 600D gas chromatograph equipped with a hydrogen- flame ionization detector. The column used for all analyses consisted‘ of a l m x 3 mm (0D) stainless steel column packed with Porapak N, 100-120 mesh (Anspec, Ann Arbor, Michigan). The oven was not heated though the operating temperature stabilized at 45 C. Carrier gas flow (high purity N2) was approximately 25 ml/min. Compressed air, at a flow rate of 300 ml/min, and H2, at a flow rate of 25 ml/min, were used to operate the flame detector. Quantities of ethylene produced were determined from a standard curve relating peak height to quantity of ethylene which was prepared using the ethylene standards. Bacterial Cultures Azotobacter vinelandii, ATCC no. 12837, A. chroococcum, ATCC no. 7499, A. agilis, ATCC no.-12838, and two strains of A. chroococcum isolated from Wintergreen Lake were used in this study. Correlation of Ultrastructure with Nitrogen Fixation Azotobacter vinelandii, ATCC no. 12837, or A. chroococcum, ATCC no. 7499, was. grown in each of two 250 ml Erlenmeyer flasks. One flask contained 50 ml of Burk's nitrogen free buffer plus 1% glucose (47), and the other, 50 ml of Burk's buffer plus 1% glucose supplemented with 0.25% NH4Cl as a source of combined nitrogen.. The flasks were incubated at room temperature (approximately 25 C) without_shaking. The cells were sampled during exponential growth and tested for acetylene reduction. At the same time, the remaining cells were collected on 7 22 mp membrane filters and washed with 3 m1 of 3% (v/v) glutaralde- hyde fixative in phosphate buffer, pH 7.2; A thin layer of 1% Noble agar was placed on the filters which were then immersed in the gluta- raldehyde fixative and kept at 4 C overnight. The filters were rinsed with cold phosphate buffer four times during the next day, sectioned into small pieces, and post-fixed overnight in 1% osmium tetroxide buffered at pH 6.1 with Veronal acetate-buffer (28) at room temperature. The fixed preparations were then soaked in 0.5% uranyl acetate solution for approximately 2 hours and dehydrated in a graded series of ethyl alcohol and prOpylene oxide. Pieces were then embedded in Epon 812 by the method of Luft (32). The blocks were cured for 24 hours at 45 C and then for 24 hours at 60 C. Thin-sections of the cured blocks were cut on an LKB III ultramicrotome (LKB Instruments, Copenhagen, Denmark) with a DuPont diamond knife and mounted on 300 mesh athene type cOpper grids.‘ Sections were doubly stained, first with 2% uranyl acetate for 10 minutes and then in lead citrate for 10 minutes. After being stained, the samples were examined with a Hitachi electron micro- scape (HU-ll) (Hitachi, Ltd., Tokyo, Japan). Isolation and Classification of Azotobacter from Wintergreen Lake Enrichment media were used to isolate Azotobacter from Wintergreen Lake. Samples were obtained aseptically (see Appendix) from Wintergreen Lake in January 1971 from depths of 5 cm, 1 m, 2m, and 3 m. A sample was collected from the mud—water interface at approximately a 5 m depth with a van Dorn sampler and placed in,a sterile container. One milliliter of each sample was pipetted into each of 4 500-ml Erlenmeyer flasks containing 100 m1 of Burk's nitrogen-free buffer plus one of the following carbon sources: glucose 0.1%, resorcinol 0.1%, mannitol 8 0.1%, or ethanol 1.0%. The flasks were incubated for 8 days at room temperature (approximately 25 C). A duplicate set of flasks was incu— bated at 12 C, the temperature of the surface water of the lake at the time of collection. Uniform turbidity was observed in the flasks incu- bated at 25 C containing 0.1% glucose or 1.0% ethanol inoculated with- the muddwater interface sample; growth did not occur in any of the other enrichment flasks. The cultures were examined microscopically for typical Azotobacter cells. One and one-half percent agar plates of Burk's buffer plus an added carbon source were used as the solid medium for streak plates. Solid medium plus 1% glucose was streaked with each of these cultures and incubated at 25 C. Isolated colonies were streaked for purity onesolid medium plus 1% glucose. The isolates, one from the ethanol enrichment and one from the glucose enrichment, were streaked along with known ATCC cultures of Azotobacter vinelandii, A. chpoococcum, and A. agilis on solid medium that contained mannitol, ethylene glycol, resorcinol, butanol, or starch. A single streak was made of each organism. Organisms growing on butanol were checked microscopically for cyst production. The isolates were assayed for their ability to reduce acetylene during growth. Growth of the Two Isolates of Azotobacter chroococcum from Wintergreen Lake Under Various Partial Pressures of Oxygen The isolates of Azotobacter chroococcum were tested for growth and acetylene reduction under various partial pressures of oxygen. Eight 500 m1 side arm flasks containing Burk's nitrogen-free buffer plus 1% glucose were set up with a gas flow apparatus as seen in Figure 18. Certified oxygen standards of 0.1%, 0.95%, 5.0%, and 20.0% oxygen in nitrogen (Matheson Co.) were bubbled through the medium. The flasks were inoculated with stationary phase cells and incubated, 9 on a shaker at 25 C. The flasks were observed periodically for absorb- ance at 660 nm in a Bausch and Lomb Spectronic 20. Twice during the exponential phase the cultures were sampled and tested for acetylene reduction.‘ The samples were flushed with the same gas that flowed through the culture and quickly capped. Preparation of Cells Used in Experiments on Nitrogen Fixation in Lake, Water, Viability in Lake Water, Repression of Nitrogen Fixation by Ammonium ioni and Nitrogen Fixation During Encystment Cells of A. vineZandEi were grown in Burk's nitrogen-free buffer plus 1% glucose at 25 C. Growth was followed by direct microscopic counting in a Petroff-Hausser bacteria counter and by turbidity measure- ments at a wave length of 660 nm in a Bausch and Lomb Spectronic 20 spectrophotometer (Bausch and Lomb, Rochester, New York). Cells in the late exponential phase of growth (about 18 hr) were harvested by centrifugation at 4 C in a RC-ZB Sorvall refrigerated centrifuge at 3000 x g for 20 minutes, washed with.water, and suspended in the appropriate solutions. Preparation of Bacteria-Free Lake Water Lake water was obtained from the surface of Wintergreen Lake in sterile containers. The water was passed through sterile 0.45 pm followed by sterile 0.22 um membrane filters (Millipore Corp., Bedford, Mass.). Nitrogen Fixation in Lake Water Cells suspended in sterile water were dispensed in 2-ml volumes into 12 100-m1 glass serum bottles. Six of the bottles contained 48 ml of bacteria-free lake water, and 6 contained 48 ml of Burk's nitrogen? free buffer. The final cell concentration was 2 x 109 cells/ml. 10 Glucose was added to each of the 6 bottles to final concentrations of 0, l, 10, 100, 1,000, and 10,000 ug/ml.. Nitrogen fixation was. assessed at zero time and at 3 hours. Zero and 3 hours are the times at which the samples were taken for the assay; however, the assay takes one hour. The data reflect the cell response over a 1-hour period. The amount of ethylene produced per hour at zero time was subtracted from the amount of ethylene produced at 3 hours to indicate a decrease or increase in the rate of nitrogen fixation. The amount of ethylene produced per hour in Burk's nitrogenefree buffer plus 10,000 pg glucose per milliliter was taken to be the maximum amount of fixation under optimum conditions. The ethylene produced in the other samples is calculated as a percentage of this maximum amount. Viability in Lake Water Cells were suspended in bacteria-free lake water or Burk's nitrogen-free buffer to a final concentration of 20 cells per milli- liter. At intervals of 0, 4, 8, 12, and 48 hours, samples were with- drawn and 1:4 dilutions made in sterile water blanks. Triplicate 0.2 m1 samples were plated on Burk's nitrogen-free agar (1.5%) plus 1% glucose.. Colonies were counted after the plates were incubated at 25 C for 24 hours. The generation time, g, was calculated according to the formula: g = t(log 2)/log b - log a, where a is the initial number of cells, b is the number of cells after time t, and t is the time interval between measurements. Radioisotopes Glucose-U-14C and L-leucine—U-14C were obtained from the New England Nuclear Corporation, Boston, Mass. 11 Respiration Studies Azotobacter vinelandii was grown in 100 m1 of Burk's nitrogenw free buffer plus 1% glucose plus 10 uc.glucose-U-14C (specific activity 207 uc per umole) for 18 hours. The cells were harvested by centri- fugation at 20,000 x g for 20 minutes and resuspended in triplicate 50 ml volumes of bacteria-free lake water or sterile Burk's nitrogen- free buffer in 500-ml Erlenmeyer flasks to a final concentration of about 2 x 1010 cells per milliliter. These 6 flasks were incubated at 30 C in a waterbath shaker. One-half milliliter samples were withdrawn from each flask at 30-minute intervals and measured for radioactivity in a liquid scintillation counter (Packard Tri-Carb scintillation spectrometer model 3310) using the following procedures: (1) Cells. The cell suspension was collected on 0.45 pm membrane filters (Millipore Corp., Bedford, Mass.) and washed with 1.5 m1 of nonradioactive buffer or lake water. Filters were dried and placed in 5.0 ml of toluene base scintillation fluid (12). (2) Supernatant. The filtrates were combined with the washings and assayed in scintillation vials containing 10 md of Bray's solu- tion (6). / (3) C0 . 1+CO was trapped in fluted Whatman no. 1 filter aper _2 2 P (3 x 5 cm) soaked with 0.25 ml 10% KOH. The filter paper was placed in a scintillation vial which was suspended in the 500-ml Erlenmeyer flask by means of a wire and was changed every 30 minutes. After being dried the paper was placed in 5.0 m1 of toluene base scintilla- tion fluid (12). 14002 counts-per minute are recorded as cumulative values. 12 Protein Synthesis The synthesis of protein was estimated indirectly by measuring the incorporation of L—leucine-U-14C (specific activity 306 uc/umole) into trichloroacetic_acid-insolub1e precipitates following the basic procedure of Yang and Brubaker (52). Cells, growing in Burk's nitrogen-free buffer plus 1% glucose, were harvested at 18 hours by centrifugation at 20,000 x g in an RC-2B refrigerated centrifuge. The cells were resuspended in fresh bacteria—free lake water or Burk's nitrogen—free buffer to which L-leucine-U-l4C was added at a concentration of 5 uc/ml. One-half milliliter samples were removed at 3-hour intervals from the cultures, mixed with 0.5 ml of cold 10% trichloroacetic acid (TCA), incubated for 30 minutes at 5 C, and then.collected on 0.45 um membrane filters. The filters were washed 3 times with 5.0 m1 of 5% cold TCA containing 5 uM/ml nonradioactive leucine to displace any l4C-leucine bound to the precipitate. The filters were dried, put into 5.0 ml toluene base scintillation fluid, and counted. Amount of Glucose in Lake Water The Hobbie4Wright technique (49), using high specific activity glucose-U-IAC and based on MichaeliséMenten_enzyme kinetics, was employed to estimate the concentration of glucose in Wintergreen Lake. To 25dml quantities of nonfiltered lake water in 100-ml glass serum bottles were added various amounts of glucose-U-lAC to give final concentrations of 5, 15, 30, and 45 ug glucose/liter. The bot- tles were capped with rubber serum caps and incubated in the lake (see Appendix) for 1 hour. Blanks were prepared by adding 45 ug glucose~U-14C/ liter immediately followed by Iz-KI (Lugol's solution containing 1 g 13 iodine, 2 g KI, and 300 ml distilled water) to inhibit metabolic activity. At the end of the incubation period the samples were fixed with 1 m1 of the IZ-KI solution. Cells were collected on 0.22 pm membrane filters, placed in 15 m1 Bray's solution (6) and counted in a scintillation counter. Results were analyzed graphically to determine the maximum glucose concentration. Repression of Nitrogen Fixation by Ammonium Ion Cells were added to a final concentration of 2 x 109 cells/m1 to each of 5 500-ml Erlenmeyer flasks containing 50 ml of Burk's nitrogen- free buffer plus 1% glucose and ammonium in the following concentra- tions: 0, 26, 52, 129, and 971 mg/liter. Samples were removed from the flasks at intervals of 0, 1/2, 1, 2, 3, and 4 hours and assayed for acetylene reduction. Cells were also added to a final concentration of 5 x 109 cells/ml to Burk's nitrogen-free buffer plus 1% glucose. Eight and seven.tenths milliliters of the cell suspension were then placed in 26 ml serum bottles and assayed for ethylene production with time. Fifty minutes, after the acetylene was injected, ammonium was injected into the vials. Ethylene production with time continued to be monitored. Triplicate vials were prepared with ammonium concentrations of 0, 3.37, 33.7, and 337.0 mg/liter. Water was added to the control. Nitrggen Fixation During Growth Stationary phase cells of A. vinelandii and the 2 isolates of A. chroococcum were inoculated into 500~ml Erlenmeyer flasks containing 200 m1 of Burk's nitrogen-free buffer plus 1% glucose to concentrations of 4 x 109 cells/ml for A. vinelandii and 17x 109 cells/m1 for each of the A. chrcococcum isolates. Cells were grown on a rotary shaker 14 (200 rev/min) at 25 C. Growth was followed by direct microscopic counting inAa Petroff-Hausser counting chamber and turbidometrically using a Bausch and Lomb Spectronic 20, wave length 660 nm. Samples were taken periodically and assayed for acetylene reduction. Cells of the A. chroococcum strain isolated from the glucose enrichment were also inoculated into each of 3 500-m1 Erlenmeyer flasks containing 200 ml of Burk's buffer plus 1% glucose. The final concentration of cells was about 2 x 109/m1. The ethylene produced was assessed every 2 hours. When the rate of ethylene production approached zero, cells in_one flask were centrifuged, washed with sterile water, and resuspended in fresh medium. Nitrogen Fixation During Encystment Cells grown in Burk's nitrogen-free buffer plus 1% glucose were harvested at 18 hours and suspended into each of 2 500-ml Erlenmeyer flasks containing 50 m1 of Burk's buffer plus 1% glucose or Burk's buffer plus 0.2% B-hydroxybutyric acid (BHB). Samples were taken periodically and checked.for absorbance at 660 nm and for nitrogen fixation. Most Probable Numbers (MPN) of Azotobacter in Wintergreen Lake Water samples were collected from the surface of Wintergreen Lake over its deepest area. One-milliliter amounts of lake water were diluted with sterile distilled water and inoculated into 5 tubes of Burk's nitrogen-free buffer plus 1% glucose. After incubation at 25 C for 10 days, the tubes were examined macrosc0pically for the presence of characteristic Azotobacter pellicles and microscopically for Azoto- bacter cells. Most probable number estimates were determined from an MPN index (44). RESULTS The effect of incubation time on the production of ethylene was studied in order to determine the proper incubation time to use for the assay of nitrogen fixation. Results are presented in Figure 1. One hour was chosen for the length of incubation for the assay since- ethylene production was linear with time up to 75 minutes. The effect of flushing the incubation chamber, prior to incorpora- tion of acetylene, with an Ar, 02, C02 gas mixture on the amount of ethylene detected is presented in Table 1. The results indicate that approximately 3.2 times more ethylene is detected when the samples are. flushed than when they are not flushed. Table 1. The effect on acetylene reduction of flushing with an Ar, 02, and C02 gas mixture to remove N 2 Sample Hr after Avg ethylene peak height (cm) Ratio of flushed No. inoculation flushed not flushed to not flushed l 8 1.45 0.45 3.20 2 10 0.80 0.25 3.20 3 12 4.83 1.35 3.57 Nitrogen fixation in lake water was studied by suspending Azoto- bacter vinelandii in Burk's buffer and comparing the ability of the cells to fix nitrogen with that of cells suspended in lake water. Glucose was added at the indicated concentrations to both the buffer 15 16 Figure l. Ethylene production by Azotobacter vinelandii vs time. 13 I 12 . I ll ' 45/} 10 - J. 9 ‘ } A Average ethylene peak height (cm) \1 4 . 3 . 2 . 1 . v v v v j i v V I V 0 15 30 45 60 75 90 105 120 135 150 minutes 17 and the lake water to determine the effect of added glucose. Ethylene was not produced in either the Burk‘s buffer or lake water until at least 100 ug glucose/ml was added (Table 2). Even when glucose was added, the amount of ethylene produced in lake water was half the maximum amount produced in Burk's buffer plus 10,000 ug glucose/ml. Table 2. N fixation by Azotobacter vinelandii in lake water: labora- tory studies Bacteria-free lake water collected 7/10/71 4pH 8.5 Burk's nitrogen-free. buffer, pH 6.8 mumoles of mumoles of Concn of ethylene ethylene glucose produced/hr % max amt produced/hr % max amt added in from t0 to of ethylene from tO to of ethylene ug/ml t3hrs* produced/hr t3hrs* produced/hr 0 -0.03 -0.03 1 -0.03 0.0 -0.03 0.0 10 -0.04 0.0 -0.03 0.0 100 +0.46 112.2 +0.19 46.3 1,000 +0.42 102.4 +0.18 43.9 10,000 +0.41 100.0 +0.20 48.8 * .. (-) indicates a decrease in ethylene production from the initial amount observed at zero time, (+) indicates an increase in ethylene production from the initial amount observed at zero time. For samples containing 0, 1, 10 ug glucose/m1 the ethylene peak was 2 times that of the dead control. When this same experiment was repeated 3 months later with the samples incubated in the lake, ethylene production occurred only in Burk's buffer plus 10,000 ug glucose per milliliter, 0.24 mumoles of ethylene was produced per hour. 18 Cells growing in Burk's buffer plus 1% glucose were transferred to lake water and buffer in an attempt to determine if the cells were viable and if they were growing. Samples were taken periodically and viable counts made. The generation times of 27.3 hr in Burk's buffer and 29.3 hr in lake water were calculated from the 12— and 48-hr data (Table 3). Table 3. Viability of Azotobacter vinelandii in_lake water Hours after Average no. of Generation inoculation organismster ml time in hrs 20 Burk's nitrogen-free 3’814 buffer 8 7,460 12 5,260 27.3 48 14,960 20 Sterile 4 1,014 lake water 7’014_ 12 10,506 29.3 48 23,426 * Calculations were done according to procedures outlined in the Materials and Methods section; generation time was calculated between 12 and 48 hours. Studies with 14C-glucose grown cells resuspended in lake water or in Burk's buffer were done to determine if the cells respire in lake water. Some 14C-glucose was carried over in the supernatant solution. This was rapidly depleted to a constant level after 2 hours (Figure 2). The respiration rate during the 2- to 18-hr interval after the cells were resuSpended in lake water was 1.4 x 10.7 umoles 14CO2 per hour, Figure 2. umoles umoles 1,200 1,000 800 600 400 200 1,200 1 ,000 900 800 700 600 500 400 300 200 100 19 Respiration of cells in lake water and in Burk's buffer. ‘ A. Respiration in lake water /0 C0 released . 2 i D D n ' ' 1 1 1 D t) o o o O o o i o 14C glucose incor— porated into cells ‘ 14C glucose in super- . v v r ‘v v . - natant solution 0 1.0 2.0 3.0 4.0 18.0 hours . B. Respiration in Burk's buffer . . . . . 9 CO2 released . 9 .. D 1 9 \ J O O o 0 o I O 14C glucose incor- 0 G. '( porated into cells D 14C glucose in super- : v I . natant SOlUtIOI’l 1.0 2.0 3.0 4.0 18.0 hours 20 compared to 1.2 x 10-7 umoles 14C02 per hour in Burk's buffer (Table 4). The protein synthesizing system was found to be active in cells suspended in lake water (Figure 3). The cells exhibit a greater rate of protein synthesis in lake water than in Burk's buffer. The rate of leucine incorporation was stimulated by the addition of 2 mg/ml glucose to lake water or to Burk's buffer. The rate of protein synthesis in lake water was greater than in Burk's buffer with and without glucose. The concentration of glucose in the lake was estimated using the kinetic analysis described by Allen (2) (Table 5). A graphical analysis of the data (Figure 4) indicates that the maximum concentra- tion of glucose in Wintergreen Lake is 13.5 ug/liter. Preliminary studies were done to determine if a correlation could be made between the ultrastructure of Azotobacter and nitrogen fixa- tion. The results (Table 6) indicate ethylene was produced in cultures without NH4C1 and was not produced in cultures with NH Cl. Electron 4 micrographs of the organisms can be seen in Figures 5 through 8. Figures 5 and 7 are of thin sections of whole cells of Azotobacter grown with N2, and Figures 6 and 8 are of cells grown with ammonia. Little dif- ference is observed in the internal membrane network in the cells grown with and without NHaCl. Ammonia nitrogen.has been reported to repress nitrogen fixation. in Azotobacter (39). In the present study, as little as 26 mg ammonia nitrogen per liter of Burk's nitrogen-free buffer plus 1% glucose was found to inhibit nitrogen fixation (Figure 9). Higher concentrations of ammonia nitrogen showed the same effect. The effect of ammonia nitrogen on ethylene production with time was studied (Figure 10). Ethylene production was gradually reduced after injection of NH4C1. 21. Table 4. Respiration studies of Azotobacter vinelandii in lake water Hours after inoculation 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 18.0 umoles 14Cglucose incorporated into cells x 108 Bacteria- free lake 260 280 390 410 390 410 390 400 390 360 water Burk's buffer 290 280 360 340 350 370 320 370 370 360 14 8 umoles C glucose in the supernatant solution x 10 Bacteria- free lake 520 300 90 60 50 50 50 60 60 60 water Burk's buffer 720 310 140 60 60 50 50 50 50 50 umoles 14CO2 released x 108 Bacteria- free lake 130 420 810 930 960 980 1000 1010 1160 water Burk's buffer 120 380 720 930 1020 1050 1080 1090 1140 The rate of increase in respiration from.2 to 18 hours in lake water is 1.4 x 10‘7 umoles 14C02/hr; and in Burk's buffer, it is 1.3 x 10"7 umoles COZ/hr. 4 pmoles leucine incorporated into TCA insoluble precipitates x 10 Figure 3. .589 1 .442 a .294. 22 Protein synthesis in lake water vs buffer. KEY: O lake water + 0.2% glucose . lake water A buffer + 0.2% glucose hours ‘ buffer C O —A A A O A 0 ‘—1I ' V V V i ' 4 U 3 6 9 12 15 18 21 24 23 Table 5. Uptake of radioactive glucose by a natural lake pOpulation C, counts/ min from u, duantity C, radio- luc 14C of 4C activity of (corrected added to t, incuba- filtered A, added for machine the sample tion time organisms in substrate efficiency in ug/l in hours counts per min* Cut/c in ug/l 177,600 5 1 29,831 29.78 5 (85% effi- ciency) 15 1 61,121 43.59 15 30 1 79,3281 67.16 30 45 1 82,665 96.68 45 Symbols are those used by Allen (2).: * Counts are corrected for blank and quenching. Table 6. N fixation by Azotobacter vineZandEi and Azotobacter cgroococcum in medium with and without NH4C1 mumoles ethylene produced/hr . Organism Without NHqC1 With NH4 C1 Azotobacter vinelandii 47 0.06 Azotobacter chnoococcum 36 0.07 Figure 4. 24 Graphical analysis of bacterial uptake at low substrate concentrations following Michaelis-Menten enzyme kinetics. Plot of Cut/c against increasing substrate concentrations (8), illustrating derivation of 1) maximum natural substrate concentrations (Kt + Sn) as ug/l; 2) maximum velocity of bacterial uptake (V) as pg per liter per hour; and 3) turn- over time for substrate regeneration (Tr) as hours. 100 1 90 ‘ y = mx + b Cut/c = A/Vt + (Kt + Sn)/Vt 80 . 70 ‘ 0 510 e = 1/V = 1.53 60 ‘ p t 1 l Vt = 0.65 pg 1. hr" 50 ‘ 40 ‘ / (Kt + Sn)/vt = Sn/vt = Tt = 22 hrs —15 —10 -5 0 5 10 15 20 25 30 35 40 45 50 [A] (added substrate) ug/l 25 Figure 5. Ultrastructure of Azotobacter vinelandii grown with N2 as the nitrogen source. Total magnification is 87,000X. Bar represents_0.2'um. Figure 6. Ultrastructure of Azotobacter vinelandii grownwith NH4C1 as the nitrogen source. Total magnification is 100,000X. Bar represents 0.2 pm. 27 , l, , , , ,i';,e Figure 7. Ultrastructure of Azotobacter chrvoooacum grown with N2 as the nitrogen source. Total magnification is 34,500X.. Bar represents 0.5 pm. Figure 8. Ultrastructure of Asotobaater chrooooccum grown with NH 01 as the nitrogen source. Total magnification is 26,000X. Bar represents 1 pm. 10 mpmoles ethylene per hour per bacterium x 10 29 Figure 9. Repression of ethylene production in Azotobacter vinelandii by ammonia nitrogen. q .14 . “z I .13 i .10 ‘ , O O .08 “ KEY: 0 control, water 26 mg ammonium per liter 0 O . w 0 0.5 1.0 2.0 3.0 4.0 hours after inoculation 30 Figure 10. The effect of NH + on ethylene production by Azotobacter vineZandii with time. ethylene peak height (cm) 30 ‘ KEY: + C) no NH4 + . 3.37 mg NH4 /1 A 33.70 mg NH4+/l ‘ 337.00 mg NH4+I1 . ./ / I N114+ added at this point f ‘ r V V V t V ' V V i 20 40 60 80 100 120 140 160 170 180 190 200 minutes after acetylene was added 31 Nitrogen fixation during the growth of Azotobacter vinelandii in laboratory cultures was studied (Figure 11). Ethylene production begins early in the exponential phase and increases until about the middle of the exponential phase, at which time the amount of ethylene produced per cell begins to decrease. The amount of ethylene produced reaches zero at the beginning of the stationary phase. Ethylene production during the growth of the Azotobacter chroococcum strain isolated from the glucose enrichment was studied. When the rate of ethylene production approached zero, cells in one flask were centri- fuged, washed with sterile water, and resuspended in fresh medium. The absorbance continued to increase while ethylene production decreased (Figure 12). Cyst formation was induced inquotobacter vinelandii by 0.2% B-hydroxybutyric acid. Nitrogen fixation during induced encystment decreased to an almost undetectable amount 1 hour after introduction into 0.2% BHB (Figure 13). Most probable numbers of Azotobacter in Wintergreen Lake were determined in July 1971 using an MPN index.‘ The results (Table 7) indicate that there is 1 Azotobacter cell in 2 m1 of lake water. Two isolates with Azotobacter morphology were obtained from enrichment cultures of a muddwater interface sample from Wintergreen Lake. The one strain that grew in ethanol produced yellow, raised, circular, gummy colonies on solid medium plus 1% glucose. The other isolate, from the glucose medium, produced slimy, spreading colonies which turned brown with age. The results of the utilization by Azo— tobacter of various carbohydrates are presented in Table 8. Azoto— bacter vinelandii, A. chroococcum; and both isolates formed cysts on bacteria per m1 No. Figure 11. 1x1010 0.05 O O 32 O M absorbance CO I I ethylene produced \J 5 10 rvvvvvijvvv'vrvv'vVijTfl—V 15 20 25 46 Ethylene production during growth of Azotobacter vine- Zandii in Burk's buffer plus 1% glucose. 10 mumoles of ethylene produced per hour per cell x 10 33 Figure 12. The effect of fresh medium on ethylene produced. log absorbance 660 nm Key: C) absorbance of control culture . ethylene produced in control culture [] absorbance in culture resuspended in fresh medium at 17 hr ID ethylene produced in culture resuspended in fresh medium at 17 hr [3 absorbance in culture resuspended in fresh medium at 23 hr ll ethylene produced in culture resuspended in fresh medium at 23 hr - 6 P I . 5 1 D ‘ D I D . D . 0 . /\ 4 E ' u . I . - ’ u b .1: on ' H ‘ I Q) , .o P 3 'fi I- m b D. 0.1 - . 8 , m ' r-4 1 D >\ 1 ’ .5 4' ‘ ‘ . 2 Q) c 4‘ D d ‘ b ‘ P V :-l N \V ' p 0'01 vvvv'vvvvrvvvvvvv"v"'*V""V""If"'v""v'Vr'v‘r" 0 0 5 10 15 20 25 30 35 40 45 50 hours 34 Figure 13. N2 fixation during encystment 40. D .23" 0 cells \ 10 \ O v-I H Q) O. H :3 O .5 H G.) I O. 'U (U o q :3 "U 8 o. 20 -I Q) C: m 4 H >5 .6 U 0 1 0) Q3 '8 B 4 1 E 10.. I cells plus 0.2% BHB 2. — _ — _ v i ' I 0 0.5 1.0 1.5 2.0 2.5 Hours after inoculation 35 Table 7. Most probable numbers of Azotobacter in Wintergreen Lake No.of tubes giving positive reactions Combination of out of 5 tubes positives MPN index per 100 ml dilution 100 101 102 103 104 2 0 0 0 0 2-0-0* 50 Table 8. Utilization of various carbon sources by Azotobacter Isolate Isolate A A. A. from from Carbon source vinelandii ohroococcum agilis glucose ethanol mannitol +4 : -|_- i ethylene glycol +1 i i i resorcinol +1 butanol +3, +2 +3 +3 starch _ +4 +4 1'. +4 +2 Growth was scored as +1 to +4 with i being barely visible growth. 36 butanol. Both isolates appeared to be A. chrooooccum. Figures 14 and 15 show the pattern of ethylene production during growth of the isolated strains of Azotobacter. Ethylene production begins in the late lag phase and increases until about the middle of the exponential phase, at_which time the amount of ethylene produced begins to decrease. The 2 isolates of A. chroococcum from Wintergreen Lake were tested for growth and acetylene reduction under various partial pressures of oxygen. The A. chroocoocum strain isolated on glucose grew equally well in 20, 5, and 0.95% oxygen atmospheres (Figure 16). The amount of ethylene produced increased in the samples grown in the 0.95% and 0.1% oxygen atmospheres (Table 9). The strain isolated on ethanol showed a different pattern of growth. It grew best under an atmosphere of 5.0% oxygen (Figure 17). Ethylene produced increased in the 20% and 0.1% oxygen atmospheres (Table 9). Table 9. Acetylene reduction during the growth of 2 strains of Azotobacter chroocoooum under various partial pressures of oxygen umoles ethylene/absorbance unit 660 nm Azotobacter chrooooccum isolated on glucose Azotobacter chroocoocum isolated on ethanol. % oxygen 24 hr 33 hr 24 hr 33 hr 20 0.316 0.037 0.328 0.415 5 0.045 0.037 0.037 0.031 0.95 0.015 0.030 0.030 0.030 0.1 0.033 0.028 0.031 0.035 37 Figure 14. N fixation during growth of Azotobacter chroocoocum strain 1 isolated from an ethanol enrichment. ’l.l pl.05 ~l.0 1.0 u _ 0.95 I d u . 9 " no N » .85 .i O 4.) - .8 '2 1 o D . .75 “g - .8 V absorbance _ .7 s5 0 U) .D N b .65 u g j - .60 g . Q) 8 3 0 ~ .55 g 8 . .. .o O H I . g E: o 5 .2: m s z 00 . .45 -fi 0 m A ‘ m C) . .4 '3 E . .35 0.. -/O c ' .3 4 ’ .25 . D ‘I O / ethylene produced ' ‘2 1 ' .15 l O 4 ’ .l D ' .05 0.001 4&1 - . j 3 v . - 10 20 30 40 50 60 70 0 Hours after inoculation Figure 15. N sgrain 2 isolated from a glucose enrichment. Log absorbance 660 nm 1.0 0.1 0.01 0.001 I ALL A 38 fixation during growth of Azotobacter chroococcum CID G O V ab sorbance ' 2.070 - 1.863 _ 1.656 , 1.449 '1.242 1.035 I P .828 . .621 I .414 ' .207 IO . ‘0 j b : G 0 ‘ J. .‘J ethylene produced . D I |._ . 10 20 30 40 50 60 70 Hours after inoculation d umoles ethvlene per absorbance unit 39 Figure 16. Growth of Azotobacter chroococcum isolated on glucose Log absorbance 660 nm 0 1.0 0.1 .01 under various partial pressures of oxygen. d i J 4 d 1 D d D KEY: ‘ D 20.07. 02 j ' 0 5.0202 4 7.; A 0.95% 02 q .9 p 0 0.1020 A 2 Q D ' u l v—VT1""""'"V'VV'VVVV'VvvvjvvvV'Vvvjfifijvvvv 0 5 10 15 20 25 30 35 40 45 Hours 40 Figure 17. Growth of Azotobacter chroococcum isolated on ethanol under various partial pressure of oxygen. d d 0* t. I> 0' 1.0 - i A d . E ‘ ‘ C: O ‘ O 1 ‘ I :8 a Q) I U a v m .0 H o m '3 D 00 KEY. 3 C) 5.0% 02 . . A 0.954 02 D 20.0% 02 0 0.174 0 2 1 1 0.01 VVV'IVVVVVV'VTtvv*1I"Vvvvvv-t"'vr'"'I*"V1' 0 5 10 15 20 25 30 Hours 41 Figure 18. Gas flow apparatus used in studies of growth under various partial pressures of oxygen. source of gas \‘ “3 rubber tubing between : glass joints I — cotton in glass tube - 500 m1 side arm flask - ‘ ' fl . 'u'or‘f’. . '1'. rc‘0.,. ' _955 “if 100 m1 Burk's buffer plus "9~“‘ 1% glucose DISCUSSION Studies of cell-free extracts from various Nz-fixing systems have shown that the Nz-fixing enzyme, nitrogenase, catalyzes the reduction of acetylene to ethylene (14,20,40). The reduction of N2 to ZNH3 requires the transfer of 6 electrons, whereas the reduction; of acetylene to ethylene requires 2 electrons. Studies on the correla— tion between acetylene reduction and nitrogen fixation indicate that the ratio of moles of N2 fixed to moles of ethylene produced ranges from 3 to 4.5 (20). Stewart, Fitzgerald, and Burris (43) have shown that it is feasible to use the rate of ethylene production as an index of the rate of nitrogen fixation. This indirect technique has several advantages over the conventionallsN technique. It is: (a) relatively inexpensive, (b) easier, (c) a more sensitive method, and (d) short-term exposures allow measurements of short-term changes. Nitrogen is a competitive inhibitor of acetylene reduction (14). Flushing with a gas phase containing no nitrogen upsets the equilibrium of dissolved gases causing N dissolved in the liquid to come out of 2 solution.‘ The importance of flushing can.be seen in Table 1;-flushing causes a threefold increase in ethylene production. The main objective of this study was to determine if Azotobacter fixed nitrogen in Wintergreen Lake. Studies of nitrogen fixation by Azotobacter vinelandfii indicated that nitrogen is not being fixed either in Wintergreen Lake water or Burk's nitrogen-free buffer until a minimum of 100 ug glucose per milliliter is added (Table 2). This indicated 42 43 that an insufficient concentration of an energy source may be limiting the rate of nitrogen fixation. However, even when glucose was added, the amount of ethylene produced in lake.water compared to that in Burk's buffer appeared to be only about one-half the maximum amount found for the buffer. The amount of ethylene produced in Burk's buffer plus 10,000 pg glucose per milliliter was considered maximal, because optimal concentrations of all elements essential for growth are.present. These results_indicated that the cells were capable of reducing acetylene but that something other than a carbOn source was affecting the amount of nitrogen fixed. The presence of a combined nitrogen source or a difference in pH might have affected the amount of nitrogen,fixed. Acetylene reduction was observed only in the sample containing Burk's buffer plus 1% glucose when the experiment was done in the lake. The general methods used were the same; however, the lake water used in_the laboratory studies and in the lake studies was collected at different times, July and October, respectively. The lake water was. collected, filtered, and stored at 4 C for use in laboratory studies, whereas in the lake experiment water was collected, filtered, and immediately used. The chemical composition of the lake varies with + show an annual range of 0.005 to 2.32 4. mg/l, N02- ranges from 0.012 to 0.04 mg/l, and N03- ranges from 0.005 the time of year, levels of NH to 1.34 mg/l (33). The levels of combined nitrogen are low from. June to August and high from September to April (33). The temperature of the lake also varies. Laboratory experiments were conducted at 25 C (the temperature of the surface of the lake in midsummer), and the lake experiment was done at 15 C. The effects of temperature on nitrogen fixation have not been studied; however, 15 C is near the 44 minimum temperature for growth (26), and the amount of nitrogen fixed has been shown to be proportional to the amount of growth. The results of experiments on nitrogen fixation in lake water indicate that Azotobacter vinelandii does not fix nitrogen in Winter- green Lake. There are several explanations for the absence of nitrogen fixation in lake water. The prime explanation is that an available energy source is lacking in lake water. Azotobacter vinelandii was suspended in lake water to determine if it remained viable and grew. Studies on the viability of Azotobacter vinelandii in Wintergreen Lake water indicate that, although cells transferred to either lake water or Burk's buffer show an initial increase in cell number, the cells do not actively grow in the lake water (Table 3). The early growth could be due to the carryover of some glucose when the cells were. transferred, to the utilization of storage products, or to the cells being ready to divide at the time of transfer. Because of this, the calculation of generation time of Azotobacter vinelandii in-lake water and in.Burk's buffer was based on the 12- to 48-hr interval after resuspension when the increase of cell number in Burk's buffer was not so rapid. The generation times, 29.3 hr in lake water and 27.3 hr in Burk's buffer, are not appreciably.different indicating maintenance level activity. The generation time of cells growing in Burk's buffer plus 1% glucose was 5.4 hr. This is approximately one-fifth that in lake water not supplemented with glucose. Studies-with cells grown in medium containing labeled glucose indicate that the cells respire in the lake water at about the same rate as they do in Burk's buffer, indicating maintenance-level respira- tion (Table 4). Some 14C-glucose was carried over in the supernatant solution, and this was rapidly depleted to a constant level after 2 45 hours (Figure 2). The l4C02 produced after 2 hours then indicated respiration in the new medium. The Burk's buffer is essentially a starvation medium. The respiration rate during the l6-hr interval. from 2 to 18 hours after the cells were resuspended in lake water was.l.4 x 10-7 umoles 14COZIhr compared to 1.3 x 10.7 umoles 14COZ/hr in Burk's buffer. It must be noted that the pH of the lake water was- 8.5, whereas that of the Burk’s buffer was 6.8. At pH 8.5 dissolved carbon dioxide tends to be in the bicarbonate form. At pH 6.8 more tends to be released from solution as 002; however, significant amounts of dissolved C02 will not be released from solution until pH 6.3. To have an accurate measure of C02 produced by the cells, dissolved C0 should be driven from the solution by the addition of 2 acid. The fact that this was not-done should not alter the results. Slightly more 14002 was trapped in KOH-in the lake water sample than in the buffer sample. Since more 002 would be dissolved in lake water at pH 8.5 than in the buffer at pH 6.5, the addition of acid would have only enhanced the amount ofMCO2 trapped in the lake water sample. This would still remain above that observed in the buffer sample. By continually removing C02 in the KGB trap, the equilibrium is forced toward C02 formation. If«the cells release 1.4 x 10.7 umoles of C02/hr/lO10 cells in lake water, the amount of energy available to the cells can be calculated. In the citric acid cycle, 38 ATP molecules are produced from 1 molecule of glucose.which is completely oxidized to 6 CO2 and water.. This means that if l.4~x 10-7 umoles of 002 were detected, 8.74 x 10.7 umoles of ATP were produced. Four ATP are required for the transfer of one electron pair. This means that 6 umoles of ATP are required per umole NH3 pro- duced, or 4 umoles ATP are required per uncle of ethylene produced. 46 If the entire amount of ATP produced were to be used in nitrogen. fixation, the maximum amount of NH3 formed would be 1.46 x 10-7 umoles; or, in the acetylene reduction technique, 2.19 x 10"7 umoles of ethylene would be formed. These low quantities of NH or ethylene 3 indicate that the cells do not have the energy required for nitrogen. fixation. It should be noted that since acid was not added to the cell suspension to drive off dissolved C02, the estimate of CO2 evolved may be low. The protein-synthesizing system.was still active in cells suspended in lake water (Figure 3). That the cells exhibited a greater rate of protein synthesis in lake water was indicated by greater incorpora- tion of L-leucine-U-14C into cold TCA-insoluble precipitates. Since leucine is one of the common amino acids found in protein, it is probably immediately incorporated into leucine tRNA.which then is used in protein synthesis. The rate of leucine incorporation was stimulated by the addition of 2 mg glucose/ml of lake water or Burk's buffer. However, the rate of protein synthesis in lake water remains greater than in Burk's buffer whether or not glucose was added. This indicates that there was something in lake water which promoted leucine incorporation; the lake water does have some available energy sources_and a source of nitrogen NBA, N02, and N03 (33), which the buffer does not have. An attempt was made to determine the amount of glucose in lake water using the kinetic analysis of Hobbie and Wright (49) of 14C- glucose uptake by a natural population. The kinetic analysis is based on MichaeliseMenten kinetics. When uptake of a solute is medi- ated by a transport system located at the cell membrane, the rate of uptake is describable in terms of MichaeliséMenten kinetics (25). 47 According to Wright and Hobbie (50), the measurable uptake of glucose at close to natural levels of concentration always follows Michaelis- Menten kinetics. The original equation derived by Michaelis and Menten is: V ' K:(iS (A) In terms of uptake transport systems, v is the velocity at a given substrate concentration S; V is the maximum velocity attained when substrate saturation of the uptake sites occurs; Km, the Michaelis constant, is by definition the substrate concentration when the velocity v is exactly oneehalf the maximum velocity V. To avoid con- fusion, the Michaelis constant is called Kt’ the transport constant. Equation (A) may be rearranged to give the Lineweaver-Burk equa- tion, derived by taking the inverse of equation (A) and multiplying both sides by S: S/v = Kt/V + S/V (B) By plotting S/v against S, a straight line is usually obtained. The linearity of the plot reflects how well the increase in velocity of the reaction in question corresponds to substrate concentration in the manner required by the Michaelis—Menten equation. Once one knows if the system follows Michaelis-Menten kinetics, the Lineweaver-Burk plot can be used to evaluate Vmax and Kt.- The slope is l/V, the inter- cept is Kt/V’ and the extrapolated line intercepts the abscissa at -Kt. When using radioactive material to measure the uptake kinetics, the velocity of uptake v (mg liter.l hr-l) at a given substrate concen- tration is given by the formula from Parsons and Strickland (37): 48 v - cf(Sn + A)/Cut (C) In this equation, c is the radioactivity of the filtered organisms (counts/min); Sn is the concentration (mg/liter) of a given substrate present in the natural sample; A the concentration (mg/liter) of added substrate; C the count/min from 1 uc 140 in the counting assembly used; u the number of microcuries added to the sample bottle; and t is-the incubation time (hr). In-this present analysis f, a factor to correct for isotope discrimination, is neglected because it is unknown. Equations (B) and (C) may be combined in an equation that describes the uptake kinetics of natural plankton when the natural substrate is unknown (49). The substrate concentration 8 in equation (B) is really the unknown plus the added substrate, or (Sn + A). Equation (C) is rearranged so that: (Sn + A)/v - Cut/c (D) This, substituted into equation (B) gives. Cut/c = (K.t + Sh)/V + A/V (E) With this equation, the data from uptake measurements from plankton at several low substrate concentrations are plotted as Cut/c versus [A] (Figure 4), giving the values for (Kt + Sn) and V. The intercept of the plot is actually a measure of the turnover time for the substrate due to uptake by means of a transport system (2). This intercept, obtained by extrapolation, is the point where A - O. From equations (D) and (E): Cut/c - Sn/vt a (Kt + Sn)/V = Tt (F) 49 Tt is the time (hr) required for the substrate to be entirely removed by the natural population. A constant rate of removal and continual regeneration are assumed. This indirect method for determining natural substrate concentra- tion is dependent on 4 assumptions:. (a) that isotOpic discrimination is minimal, (b) that the amount of c—14 excreted is insignificant, (c) that C-l4 is actually being incorporated into macromolecules sub- sequent_to uptake, and (d) that the natural substrate concentration is much smaller than the concentration of added substrate. The determination of natural substrate concentration is based on extrapo- lation which may not be valid. The method is based on.the uptake and not growth; consequently, the C-14 glucose may be taken into the cell and stored, it may be incorporated into macromolecules, it may be respired and lost as 14C02, or it may be adsorbed to cell surfaces. The natural pOpulation of organisms may respond to the added glucose by a switch in enzyme activity. Catabolite repression may occur; the increase in the concentration of glucose could shut off the uptake of other carbon sources and show an increase in the uptake of glucose. This would affect the observed velocity of uptake-and slope of the line which is extrapolated to obtain the concentration of natural sub- strate in the lake. An unlabeled control cannot be used. The results (Figure 4) indicate that the system follows Michaelis- Menten kinetics. The maximum level of glucose present in the lake is calculated to be 13.5 ug/liter. This is not enough to promote growth and nitrogen fixation by Azotobactar (see studies on viability and nitrogen fixation in lake water). Glucose is not the only carbon source in the lake. Manny-(33) reports that the annual range of dis- solved carbon in Wintergreen Lake~varies from 6 to 9.1 mg l-1 at a 50 l m depth. This dissolved carbon could include acetate, peptides, and organic acids. We know from the studies of viability, respiration, and protein synthesis in lake water that the cells have essentially a maintenance level metabolism. Suitable energy sources appear to be limiting. Because nitrogen fixation requires energy, and the energy is not available, nitrogen fixation is not detected. When glucose was added to lake water, the cells were able to reduce acetylene. However, even when glucose was added, the amount of ethylene produced in lake water compared to that in Burk's buffer appeared only about one-half the maximum.amount found for the buffer. This indicated that something, other than an energy source, was affect- ing the amount of nitrogen fixed. It has been shown that combined nitrogen represses nitrogen.fixation in Azotobacter (8,10,21,39). Vukhur and Iwasaki (39) report that ammonia nitrogen at a concentration as low as 410 mg ammonium per liter will inhibit nitrogen fixation in a synthesis medium. Using the sensitive acetylene reduction technique, it appears that as little as 26 mg ammonia nitrogen per liter will inhibit nitrogen fixation in Burk's buffer plus 1% glucose (Figure 9). Ethylene production with time gradually decreases when ammonium is added to a cell suspension producing ethylene (Figure 10). It-is postulated that the ammonium prevents the synthesis of nitrogenase, and the existing nitrogenase was diluted out (42). Studies of the ammonia concentration in Wintergreen Lake (33) indicate that ammonia concentrations have an annual range-of 0.005 to 2.32 mg/liter at a l m depth. Since the surface waters are fairly well mixed, the concen- tration in the surface layers should be uniform. There were also detectable amounts of nitrite and nitrate in.the lake water (33). 51 Nitrite and nitrate will also be utilized by Azotobacter in preference to atmospheric nitrogen (10,21,39). The amounts of ammonia, nitrite, and nitrate in the lake water fluctuated throughout the year. They were lowest from June through August, a time when there was an increased number of organisms which utilize the combined nitrogen for growth (33). Since the laboratory experiments on nitrogen fixation in lake water were done in July, it is highly probable that the levels of combined nitrogen-in the lake water were low, NH4 0.005 mg/l, NO 0.012 mg/l, 2 3 0.005 mg/l (33). In October, the combined nitrogen levels are NO high; ammonia concentration may be as high as 2.32 mg/l, which might account for why no nitrogen was fixed by cells suspended in lake water at that time . Studies by Oppenheim and Marcus (35) on the correlation of the ultrastructure of Azotobacter vinslandii with nitrogen source for growth indicated that Azotobacter synthesizes an extensive, internal, membranous network when grown under conditions requiring nitrogen fixation. When.Azot0bacter grows with fixed nitrogen, i.e., ammonia or amino acids, very slight quantities of internal membrane were found. These were concentrated mainly about the cell periphery. A correlation between the ultrastructure of Azotobacter‘with nitrogen fixation would have been a useful tool in indicating the capacity of Azoto- bacter to fix nitrogen. When cells were grown with N2 and ammonia for nitrogen sources, the same internal membrane network was visible' in all cells (Figures 5 through 8) regardless of the nitrogen source in the medium. These results differed from those obtained by Oppenheim and Marcus. There were 2 basic differences in the procedure: the cells were not aerated on a shaker, and the cells were double fixed with glutaraldehyde and osmium tetroxide. The double fixation process 52 allows better preservation than with simple fixation in 0804. There- fore, it seems probable that the differences in the internal membrane network observed by Oppenheim and Marcus was due to the aeration rather than to the nitrogen source for growth. The ultrastructure of the cell does not appear to be a good indicator of whether the cells are fixing nitrogen or not. The difference in pH between lake water, pH 8.5, and Burk's buffer, pH 6.8, may make a difference in the amount of nitrogen fixation observed; the nitrogenase enzyme Operates over a fairly narrow pH range near pH 7.0, with fixation falling off markedly above and below pH 6.5-7.0 (9). To see if the pH does affect nitrogen fixation, the pH of the Burk's buffer should be adjusted to that of the lake water and the nitrogen fixation tested. The pattern of acetylene reduction during growth of cells in a normal laboratory culture was studied. Ethylene production began in the late lag phase or early exponential phase of growth. Ethylene production increased until about the middle of the exponential phase, at which time the amount of ethylene produced per cell began to decrease (Figures ll, 14 and 15). One would expect that ethylene production would continue throughout the exponential stage at the same rate_as the cells were actively metabolizing and in need of nitrogen. The possibility existed that the cells accumulated waste products in the medium.would inhibit the process; accumulation of nitrogen inside the cell would show the same inhibition. When the rate of ethylene prO? duction in a culture approached zero, cells were washed and resuspended in fresh medium. The absorbance continued to increase while the ethylene production decreased (Figure 12). This indicated that the decrease in the ethylene production was not due to waste_product 53 buildup but, rather, to the physiological state of the cell. The culture is nonsynchronous; the cells are not all in the same physio- logical state. For example, cysts may have been present in the late exponential phase. Cyst formation in Azotobacter is induced by 0.02% B-hydroxybutyric acid (30). Studies of nitrogen fixation during induced cyst formation indicate that nitrogen fixation rapidly decreases to an almost unde- tectable amount 1 hour after introduction into 0.2% BHB (Figure 13). This indicates that cysts do not fix nitrogen. Since BHB is not normally found in the natural environment, the cyst, induced by BHB, may be a laboratory phenomenon. Layne and Jehnson (29) indicate that cysts may be induced by a lack of or suboptimal concentration of several minerals. Cysts formed under mineral deprivation do not have the typical ultrastructure of cysts formed in BHB. Nitrogen fixation by cysts formed under these conditions has not been investigated. Mineral deficiency in the natural environment might cause_cysts to be formed. Two isolates with Azotobacter morphology were obtained from Wintergreen Lake. The morphology, carbon utilization, acetylene reduction, pigment and cyst production indicated that the isolates areboth strains of Azotobacter chroococcum according to the classifi- cation of Azotobacter by Norris and Chapman (34) and VOets and Dedeken (45). The pattern of ethylene production during growth was the same for them as that observed for A. vinelandii. The sample from.which the two strains of A. chroococcum were isolated was not obtained aseptically introducing the possibility that the organisms were not indigenous to the lake. The isolates did not grow as rapidly or as well as A. vinelandii in the Burk's nitrogen-free.buffer plus 1% glucose. 54 Therefore, A. vinelandii was used in the major studies on nitrogen fixation. The isolates of Azotobacter vineZandii. were tested for growth and acetylene reduction under various partial pressures of oxygen. The results (Figures 16 and 17) indicated that the 2 strains had different oxygen requirements. The A. chroococcum strain isolated on glucose grew equally well in 20, 5, and 0.95% oxygen atmospheres. The strain isolated on ethanol grew best under an atmosphere of 5.0% oxygen. The ethylene produced (Table 9) was only measured at 2 points and may not be a valid indication of what actually occurred. These studies indicate that Azotobacter vinelandii does not sig- nificantly contribute to nitrogen fixation in Wintergreen Lake water. It does not fix nitrogen in lake water. Energy sources appear to be limiting. Azotobacter vinelandii exhibits a very low metabolic rate, low respiration rate and a low growth rate in lake water. Most probable number studies (Table 8) indicated that Azotobacter are present in very low numbers in the lake, about 1 organism per 2 milli- liters of lake water. This is only an indication of the number of azotobacters of any species. It is important to note that the Azoto- bacter vinelandii used in these studies was not indigenous to the lake. LITERATURE CITED 10. ll. 12. 13. LITERATURE CITED Alexander, M. 1971. Microbial ecology. John Wiley & Sons Inc. New York, p. 113, 419. Allen, H. L. 1969. Chemo-organotrophic utilization of dissolved organic compounds by planktic algae and bacteria in a pond. Int. Revue ges Hydrobiol. 54: 1-33. Beijerinck, M. W., and van Delden, A. 1902. Ueber die Assimila- tion des freien Stickstoffs durch Bakterien. Zentr. Bakt. Parasitenk., Abt. 11, 9: 3-43. Bortels, H. 1930. Molybdan.als Katalysator bei der biologischen Stickstoffbindung. Arch. Mikrobiol. 1: 333-342. Bortels, H. 1936. Entersuchungen fiber sie-Bedeutung von Molybdan, vanadium, Wolfram und andere Erdaschenstoffen fur stickstoff- bindende und andere Mikroorganism. Zentr. f. Bakt., Abt. II, 95: 193. Bray, G. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1: 279-285. Burk, D. 1934. Azotase and nitrogenase in azotobacter. Ergeb. Enzymeforsch. 3: 23-56. Burk, D., and Lineweaver, H. 1930. The influence of fixed nitrogen on Azotobacter. J. Bacteriol. 19: 389. Burris, R. H. 1966. Biological nitrogen fixation. Ann. Rev. Plant Physiol. 17: 155-184. Burris, R. H., and Wilson, P. W. 1943. Utilization of combined nitrogen by Azotobacter. J. Bacteriol. 45: 17-18. . 1946. Comparison of the metabolism of ammonia and molecular nitrogen in.Azot0bacter. J. Biol. Chem. 165: 595-598. Byfield, J. E., and Scherbaum, 0. H. 1966. A rapid radioassay technique for cellular suspensions. Anal. Biochem. 17: 434-443. den Dorren de Jong, L. E.~ 1938. Das Verhalten von Azotobacter chroococcum unter abnormen Lebensbedingungen. Arch. Mikrociol. 9: 223-252. 55 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 56 Dilworth, M. 1966. Acetylene reduction by nitrogen-fixing prepa- rations from Clostridium‘pasteurianum. Biochim. Biophys. Acta 127: 285—294. Fischer, W. K. 1948. Untersuchung zur Stammfrage bei Azotobacter chroococcum Beij. Arch. Mikrobiol. 14: 353-406. Fuller, J. E., and Rettger, L. F. 1931. The influence of com- bined nitrogen on growth and nitrogen fixation by-azotobacter. Soil Sci. 31(2): 9-234. Gainey, P. L. 1928. Sources of energy for Azotobacter, with special reference to fatty acids. Ann. Missouri Botan. Garden 15: 113-168. Greaves, J. E., and Anderson, A. 1936. Sulfur requirements of Azotobacter chroococcum. Soil Sci. 41: 197-201. Green, M., and Wilson, P. W. 1953. The utilization of nitrate nitrogen by the azotobacter. J. Gen. Microbiol. 9: 89-96. Hardy, R. W. F., Holsten, R. D., Jackson, E. K., and Burns, R. C. 1968. The acetylene-ethylene assay for N fixation: laboratory and field evaluation. Plant Physiol. 43: 1185-1207. Horner, C. K., and Allison, F. E. 1944. Utilization of fixed nitrogen by azotobacter and-influence on nitrogen fixation.' J. Bacteriol. 47: 1-14. Horner, C. K., and Burk, D. 1934. Magnesium, calcium, and iron requirements for growth of Azotobacter in free and fixed nitrogen. J. Agr. Research 48: 981-995. Horner, C. K., Burk, D., Allison, F. E., and Sherman, M. 1942. Nitrogen fixation by Azotobacter as influenced by molybdenum and vanadium. J. Agr. Research 65: 173-192. Iswaran, V., and Sundara Rao, W. V. B. 1964.~ Role of cobalt. in nitrogen fixation by Azotobacter chroococcum. Nature 203: 549. Jennings, D. H. 1963. The absorption of solutes by plant cells. Oliver and Boyd, London. p. 204.. Jensen, H. L.‘ 1954.‘ The Azotobacteriaceae. Bact. Rev. 18: 195. Johnstone, D. B. 1967. Isolation of Azotobacter insignia from fresh water. Ecology 48: 671-672. Kellenberger, E., Ryter, A., and Sechaud, J. 1958. Electron microscope study of DNA-centaining plasmas. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J. Biophys. Biochem. Cytol. 4: 671-676. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 57 Layne, J. S., and Johnson, E. J. 1964. Natural factors involved in the induction of cyst formation in Azotobacter. J. Bacteriol. 87: 684-689. Lin, L. P., and Sadoff, H. L. 1968. Encystment and polymer pro- duction by Azotobacter vineZandii in the presence of B-hydroxybutyrate. J. Bacteriol. 95: 2336-2343. Lind, C. J., and Wilson, P. W.7 1942. Nitrogen fixation by Azo- tobacter in association with other bacteria. Soil Sci. 54: 105- 111. Luft, J. M., 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol° 9: 409-414. Manny, B. A. 1971. Interaction of dissolved and particulate nitrogen in lake metabolism. A thesis submitted to Michigan. State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. p. 18,19,39,40,57. Norris, J. R., and Chapman, H. M. 1968. Classification of Azo- tobacters. In~B. M. Gi-bs and D. A. Shapton (ed) Identification methods for microbiologists, part B. Academic Press, New York. Oppenheim, J., and Marcus, L. 1970. Correlation of ultrastructure in Azotobacter vineZandii with nitrogen source for growth. J. Bacteriol. 101: 286-291. Parker, C. A.' 1954.‘ Effect of oxygen on the fixation of nitrogen by Azotobacter. Nature 173: 780-781. Parsons, T. R., and Strickland, J. D. H. 1962. On the production of particulate organic carbon by heterotrophic processes in sea water. Deep-Sea Res. 8: 211-222. Rippel, A. 1936. Eisen-, Agar- und Humuswirkung bei Azotobacter. Arch. Mikrobiol. 7: 590-597. Rubenchik, L. I.’ 1963. Azotobacter and its use in agriculture. Israel Program fer Scientific Translations, Jerusalem. Schollhorn, R., and Burris, R. H. 1966. Study of intermediates in nitrogen fixation. Federation Proc. 24: 710. Stapp, C., and Ruschmann, G. l924.* Zur Biologic von Azotobacter. Arb. biol. Reichsanstalt Land-u. Forstwirtsch, Berlin-Dahlem, B. Stewart, W. D. P. 1969. Biological and ecological aspects of nitrogen fixation by free living micro-organisms. Proc. Roy. Soc. B. 172: 367-388. Stewart, W. D. P., Fitzgerald, G. P., and Burris, R. H. 1967. In situ studies on N2 fixation using the acetylene reduction technique. PNAS 58: 2071-2078. 44. 45. 46. 47. 48. 49. 50. 51. 52. 58 Taras, M. J. [Chairman], Greenberg, A. E., Hoak, R. D., and Rand, M. C.~ 1971. Standard methods for the examination of water and waste water. 13th Edition, APHA, AWWA, WPCF. p. 673. Voets, J. P., and Dedeken, M. 1968. A physiological approach to the classification of the genus Azotobacter. International Congress for Microbiol., IX, Moscow, 1966. Wilson, P. W., and Burris, R. H. 1953. Biological nitrogen fixation, a re-appraisal. Ann. Rev. Microbiol. 7: 415-432. Wilson, P. W., and Knight, S. G. 1952. Experiments in bacterial physiology. Burgess Publishing Co., Minneapolis. Winogradsky, S. 1938. Sur la morphologie et l'oecologie des Azotobacter. Ann. inst. Pasteur 60: 351-400. Wright, R. T., and Hobbie, J. E. 1965. The uptake of organic, solutes in lake water. Limnol. Oceanogr. 10: 22-28. . 1966. Use of glucose and acetate by bacteria and algae in aquatic ecosystems. Ecology 47: 447-464. Yamagata, V., and Itano, A. 1923. Physiological study of.Azot0- bacter beijerinckii and Azotobacter vinelandii types. J. Bac- teridl. 8: 521. Yang, G. C. H., and Brubaker, R. R. 1971. Effect of Ca2+ on the synthesis of deoxyribonucleic acid in virulent and avirulent Yersinia. Infection and Immunity 3: 59-65. APPENDIX APPENDIX A Sterile Sampling Device for Lake Water and a Holder for Incubating Samples in the Lake Samples were obtained aseptically in sterile eye and ear syringes. A pasteur pipette was sealed at one end. The open end was inserted into an eye and ear syringe that had the air forced out of it. This caused a vacuum. The sampling device was wrapped in aluminum foil and. autoclaved. At the lake site, these were carefully unwrapped and placed in a_wire basket suspended by a plastic cord and weighted down- with a weight. A small wire ring was placed around the pasteur pipette to hold it upright. The sampler was lowered to the desired depth and a weight dropped down the plastic cord. This broke the pasteur pipette, and water was drawn into the bulb (Figure.19).~ A plexiglass holder was designed and built to maintain 16 serum bottles at a single depth in the lake. The holder can be lowered and maintained at any depth by adjusting the nylon cords which attach to a buoy. The holder prevents sample bottles from tilting and holds them tightly so they cannot be lost. The holder fits easily on the lap, forming a workbench during inoculation and addition of various chemical substances (Figure 20). 59 60 Figure 19. A sterile sampling device for lake water. F- plastic cord sealed._a. Pasteur pipette .9 0. “‘93. 's?‘ {'8'}? 5“". ._. 0‘. wire ring to hold pipette in place wire basket eye and ear syringe . u 0 o ' C v . 4‘ '.:O.'I;°.:0/I.‘ ' ' O a ' 0 .0 ’I 'I‘ 5 o '0_’. O... . .‘O ’ .0”- 0'. a “1. weight to fill the bulb weight dropped down the plastic cord breaks the Pasteur pipette vacuum in the syringe is released when the pipette is broken causing the lake water 61 Figure 20. Plexiglass holder for incubating samples in the lake. nylon cords tie to buoy nylon screws to hold 1“ top bars \‘=:- .“v in place \‘1' ““"i.‘\ serum bottles crews to attach weights and support cords to top View of holder $ 49 Q? 0 0 {3 support bar - _ os’fi‘Qo’a top bar in open position__q'“ 6 l u " H "I I". F.“ I “ E” H || 5" u H u _ 31293 03046 20