GROWTH, RESPIRATION, IINeEIIERGETIcs 0F NITROBACTER AeILIs IN; , THE PRESENCE OF SELECTED PESTICIDES A Thesis for the Degree of Ph;jD.. '7 MICHIGAN STATE UNIVERSITY. ‘ - , mmmeuA =: 1970 ‘ _ « - IIIIIIIIIIIII LIBRARY I H I- «Ig. 3 1293 0277 6733 . -- .i . Michigan State University This is to certify that the thesis entitled GROWTH, RESPIRATION, AND ENERGETICS OF NITROBACTER AGILIS IN THE PRESENCE OF SELECTED PESTICIDES presented by Carl L. Winely } has been accepted towards fulfillment ' of the requirements for ‘ Ph.D. degree in_m£2121_0108y 5‘ Public Health /ZJ/m( awe é,“ rMajor professor C.L. San Clemente Date 18 May 1970 0-169 OVERDUE FINES: ._.______..__. 25¢ per day per item MATERIALS: RETURNING LIBRARY to remove in book return Place records .‘c . f from circulation “MFR ....\ ‘9’” 15".? In; charge ABSTRACT GROWTH, BESPIRATION, AND ENERGETICS or W m IN THE PRESENCE or SELECTED PESTICIDES by Carl L. Winely The purpose of this study was to determine the mode of action of several pesticides on both the growth of ‘flifizgpgggg: agilis in aerated cultures and on the respiration of g. agilis cell suspensions in the Warburg respirometer. Those compounds which inhibited both growth and respiration were tested for an effect on several enzymatic activities to characterize the site of inhibition. Two pesticides (aldrin and simazine) were not inhibitory to growth of Nitgobactgr, but five compounds (CIPC, chlordane, DDD, heptachlor, and lindane) prevented growth when added to the medium at a concentration of 10 ug per ml. Whereas CIPC (110 ug/ml) and eptam (275 ug/ml) prevented nitrite oxidation by cell suspensions, the addition of DDD (500 ug/ml) and lindane (500 ug/ml) resulted in only partial inhibition of the oxidation. Heptachlor (500 ug/ml) and chlordane (500 ug/ml) also caused only partial inhibi- tion of oxidation with Nitrobagter cell suspensions, but were more toxic with cell-free extract nitrite oxidase. None of the pesticides inhibited the nitrate reductase activity of cell-free extracts, but most of them, especially heptachlor, caused some repression of cytochrome c oxidase 30t1V1tye Carl L. Winely Two of the herbicides (CIPC and eptam) exerted an uncoupling effect on the oxidative phosphorylation linked to nitrite oxidation in Nitzobgcte; agil; . Although nitrite oxidation was inhibited, the more severe effect on phos- phorylation caused decreased P/O ratios (umoles of 32F incorporation per uatom of oxygen uptake). A 50% reduction in the P/O ratio occurred at approximately 2.3 x 10'“ M CIPC and at 8 x 10'“ M eptam. A classical phosphorylation uncoupler, 2,u-dinitro- phenol (DNP), affected the oxidative phosphorylation in u. agilig in a manner similar to that observed with the herbicides. Again, oxygen uptake was inhibited, but P/O ratios were lowered because of a greater effect on phosphate esterification. A USS reduction in the P/O ratio occurred at 2.5 x 10’“ M DNP. The maximum P/O ratio obtained was 0.36. This low P/O ratio was attributed to disruption of the internal integrity of the phosphorylating particle since an active ATPase was observed when the yeast hexokinase and glucose trapping system was omitted from the reaction system. The actual modes of action of chlordane, DDD, hepta- chlor, and lindane on y. agilis were not determined, but were shown to be on biosynthetic processes of the cell rather than nitrite oxidation. In the case of the two herbicides, CIPC and eptam, uncoupling of oxidative phosphorylation in a manner ana10gous to 2," dinitrOphenol is suggested as the mode of inhibition in Nitrobacter. GROWTH, RESPIRATION, AND ENERGETICS OF NITROEACTER AGILIS IN THE PRESENCE OF SELECTED PESTICIDES By Carl L. Winely A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1970 L7 ‘- 4/17 [,I/ 1) ./ I! I... ' J ./../“'“{7« I“) DEDICATION This thesis is affectionately dedicated to my wife whose patience and encouragement made this achieve- ment possible. ACKNOWLEDGMENTS The author gratefully acknowledges the constant guidance and assistance of his major adviser, Dr. C. L. San Clemente, throughout the course of this investigation. Thanks are also extended to Dr. H. N. Costilow, Dr. H. L. Sadoff, Dr. A. B. Welcott, Dr. C. H. Cunningham, Dr. C. H. Suelter, Dr. F. J. Malveaux, A. in Garretson, W. L. Muth, and A. E. Stoekland who contributed useful suggestions, help, or encouragement which assisted in completion of this thesis. ii TABLE OF CONTENTS Page ACKNOWLEGMENTS. O O O O O O O O O O O O O O O O O O O 11 LIST OF TABLES e c e e e c e c c c e e e e c e c e e e V LIST OF FIGURES. e e e e c e e c c c e c c e e e e e 0 v1 INTRODUCTIOK e e e e e e e c e c e e e c e e c e e c e 1 LITEBAIUBE REVIEW. 0 c e c c c e e e c c c c c c c c e 3 Media and Culture TeChniqueS e e c c c c c e c 3 Inorganic Amendments to Medium . 5 Molybdenum. e c c e e e c c e e c e e 5 Yeast extract and casamino acids. 5 Acetate and formate e e c e c c c c e e e 6 BiOtlne e c e c c c e c e e c c c c e e c 6 Obligate or Facultatlve AutotrOph e e e e c e e 7 Nitrite 0X1dasec e e e e e e e c e e c c e e e 9 CYtOChromOS inv01vedc c e e e e e c c e e 9 Coupled oxidative phosphorylat tion . 10 Source of NADHZ e e c e e e c e c e c e e 11 Nitrate BOduCDasee e e e e e c c e e e e e e 13 Mode of Action of Pesticides . 13 IUSQCtiOidOBe c c c e c e c e c e e e c e 13 HerbicidGSc c e c e e c e c c e e e e c e 1“ Effect of Pesticides on Nitrification. 16 IflSOCthldCSc c e e c c c e c e e e e e c 16 Herbicides. e e e c e e c e e e e c e e e 17 MATERIALS AND METHODSe c e e c c e e e e c e e e c c c 19 PeStlcideS e c e e e e e e e e e e e e e c e c 19 IUBCCtiCldeSe c e e e e e c e c c e c c o 19 Herbicides. c e c c e e e o e e e e c e e 19 Medium 0 e e c e c e c e c c e e e e e e o c 20 GrO'th Effects 0 c c e c e c e c e e c e o e 20 Large-Scale Cultivation. e e e c e e c c e e c 21 Preparation of Cell-Free Extracts. 22 Purity of Cultures e e c e c e c c c e e e c c 23 OXLdation Of Nitrite e e e e c c e e e c e e e 23 Nitrate Reductasee e e c e c c c c e e c e e c 2“ 111 Page NADH ox1d83€e e e e c e c c e e c e e c e c e 25 SpectrOphOtometrlc assay. 0 e e e c c c c 25 Besplrometer assay. 0 c e c c c c e c e e 25 CYtOOhrome C 011(1883 c c e c e e e c c e e c 26 Oxidative Phosphorylation Coupled To Nitrite Oxidation. e e 320 e e e e e e e c e e 26 Assay by KZHPOu- P incorporation . 26 Glucose-6-phosphate dehydrogenase assay . 28 Analytical MQEhOdS e c e e c e c e e e e e e e 28 HBXOklnasee e c e e c c e e e e c e e e e 28 PrOtein e e c e c e e c c e e e e c e e e 29 O BEWLTS. O O O O O O O O O O O O O O O O O O O O O I ‘63 GrOWth Effects 0 e c e e e e e c e c 30 Oxidation Studies with Cell SuSpensions and Coll-Free EXtraOtS e c e e c c e e c e c e 30 Cells grown in ld-liter carboys . 30 Cells grown in MF-lh microferm fermentor e e c e e e c e e c c c c c c 33 Kinetics Of 011dat10nc e e e e c c e e e e e c 40 Nitrate Boduotase. e c e c e e e e e e e e c e “1 NADHZ Oxidase. e e e c e c e c c c e e e e c c an Cytochrome C Oxidase c e c e e e e c e e “8 Oxidative Phosphorylation Coupled to Nitrite OXIdBtionc e e e e e c c c c e e e e c c e e 50 Preparation of cell-free extract.