South Dakota State University Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange Electronic Theses and Dissertations 1986 Enrichment, Isolation and Characterization of Alachlor, Carbofuran, and Dicamba-degrading Bacteria Obtained from Soil Neal R. Adrian Follow this and additional works at: https://openprairie.sdstate.edu/etd Recommended Citation Adrian, Neal R., "Enrichment, Isolation and Characterization of Alachlor, Carbofuran, and Dicamba- degrading Bacteria Obtained from Soil" (1986). Electronic Theses and Dissertations. 4342. https://openprairie.sdstate.edu/etd/4342 This Thesis - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. EN RICHMENT , ISOLATION AND CHARACTERIZATION OF ALACHLOR, CARBOFURAN, AND DICAMBA -DEGRAD ING BACTER IA OBTAINED FROM SOIL BY NEAL R. ADRIAN A thesis submitted in partial fulfillment of the requirement for the degree Mas ter of Science Maj or in Microbiology South Dakota State Univers ity 1986 ENRICHMENT, ISOLATION AND CHARACTERI ZATION OF ALACHLOR, CARBOFURAN, AND DICAMBA-DEGRAD ING BACTERIA OB TAINED FROM SOIL Th is thesis is approved as a creditable and independent inv est igation by a candidate for the degree, Master of Sc i- ence, and is acceptable for meeting the thesis requi rements for this deg_ree . Acceptance of this thesis does not imply that the conclusions reached by the candidate are neces- sarily the · conclus ions of the maj or department. Dr. W. Kennedy Gauger Date Thesis Advisor .VI. • L\.UUt:::L" l.. .1.000 Date Hea d, Microbiology Dept. ACKNOWLEDGEMENTS The author wishes to express thanks and appreciation to Dr. W. Ken nedy Gauge r for his guidance, support and con­ struc tive crit icism in the preparation and editing of this manuscript . A special thanks to Dr. Duane Matthees for his support , expert ise, and laboratory facilities throughout this stud y. My time spent at South Dako ta St ate University's Mic ro­ biology Departme nt was an enj oyable and enlightening period that clarified my career go�ls . i TABLE OF CONTENTS Page ACKNOWLEDGEMEN TS . i TABLE OF CONTENTS .... ... ............... ...... ; . ii LIST OF FIGURES . 1v LIST OF TAB LES . v i INTRODUCTION . 1 Chapter 1 . • . • • . 3 LITERATURE REV IEW . 3 CHAPTER 2 ....... .......... ...... ... ..· .. 34 SCREENI NG FIELD ISO LATES FOR INCIDENTAL AND CATABOLIC PESTICIDE METABOLISM . 34 MATERIALS AND METHODS . 34 RES ULTS AND DI SCUSSION . 53 CHAPTER 3 ....· ..................................... 85 PESTIC IDE DEG RADATION BY BACTERIA ISOLATED FROM AN AGRICULTURE CHEMICAL SPILL 85 MATERIALS AND METHODS . ... .. 85 RES ULTS AND DI SCUSS ION . .. .. 93 ii CHAPTER 4 ............•....•.......•........•...... 101 ENRICHMENTS FOR MIC ROORG ANIS MS UTILIZI NG PES TICIDE S FOR CARBON, NITROGEN , OR PHOSP HORU S SOURC ES ..... ........ 101 MATERIALS AND METHODS ................. ... 101 RE S ULTS AND DI SCUSS ION .... ..... ...... 10� CONCLUSIONS . 111 LITERATURE CITED . 114 iii LIST OF FIGURES Figure Page 1 a. Screening for pesticide degradat ion by bac- teria after four weeks incubat ion ..• ....•......... 59 lb . Screening for pest icide degradation by bac- teria after four weeks incubation ............... 60 1 c. Screening for pest icide degrada tion by bac- teria after four weeks incubat ion ............... 61 2 . Deg radat ion of alachlor in complex (tryp tone­ yeast extract) , defined + yeast extract (Hutner's basal + 0.05% yeast ext ract), and def ined· (Hutne r's basal ) media .................. 64 3 • Degradat ion of ala chlor in complex (tryp tone­ yeast �xt rac t ), defined + yeast extract (Hutner's basal + 0.05% ye ast ext r act), and defined (Hutner's basal) media by St rept omyces pilosu s ............................ 65 Deg radat ion of ·al a chlo r in complex (tryp tone­ yeast extract) , defined + YE (Hutner's basal + 0.05% yeast ext ract) , and def ined (Hutner's basal) media by St reptomycete · Isolate 5 ......... 67 s: Degradat ion of ala chlo r in comple x (tryp tone­ yeast extract), defined + YE (Hutne r's basal + 0.05% yeast extract), and def ined (Hutner's basal ) media by St rept omycete Isolate 26 ........ 68 6 . Rap id plasm id DNA sc reening of Isolates using either an alkaline or polye t hylene glycol (PEG) procedure .......................... 70 7a. Alachlor degradat ion by Isolate 5 in a soil ·slu rry supplemented with ala chlor . 75 7b . Alachlor degradati�n by Isolat� 5 in a soil slu rry supplemented with ala chlor 76 iv 8a . Alachlor degradation by Isola te 5 in a soil slurry from an agricultural chemical spill 77 8b . Alachlor degradat ion by Isolate 5 in a soil slurry from an agricultural chemical spill 78 9a. Alachlor degradat ion by Isolate 5 in a soil slurry supplemented with alachlor 7 9 9b. Alachlor degradat ion by Isolate 5 in a soil slurry supplemented with alachlor 80 1 0 • Growth of two isolates obtained from Brook ings chemical spill in a basal med ium with alachlor as sole source of carbon and energy ............ 94 1 1 . Growth of two isola t es obtained from Brookings chemical spill in bas al medium with tri flu ralin as sole source of carbon and ene rgy ............. 95 1 2 • Growth-of two isola tes obtained from Brookings chemical spill in basal medium not containing an addit ional source of carb�n and ene rgy ........ 96 1 3 . Ala chlor degradat ion by two isola tes obtained from Brookings chemical sp ill ........... ......... 97 v LIST OF TABLES Tab le Page 1 . Growth of isolates on 2% unwashed and washed agar in HB broth supplement ed with various carbon and ene rgy sources .................•.. 54 2. Growth of isola tes in broth media containing various carbon and ene rgy sources 57 3 . Milligrams of mycelial biomass harvested from each culture flask following degradat ion of alachlor in three types of culture media ...... 63 4. Summary of taxonomic characteristics for alachlor-degrading bacteria ............ 84 5 . Ala chlor degradation after four weeks by eight bacteria isolated from an agricul- tural chemical spill ................... 99 6 . Enrichments performed with various pesticides added as either carbon and ene rgy, nitrogen, or phosphorus sources ....................... 105 vi INTRODUCTION The prod uction and use o � synthetic chemicals fpr pest icide control increase d dram atically after World Wa r II. Th ey were found to be highly effective , cheap to pro- duce and easy to apply. Development of these chemic als has been one of the major developments for agricul ture . In the 1970's public health risks and environme ntal damage caused by agricultural chemic als was recog nized . Problems of chronic exposure� improper waste disposal and environmental dam age developed wh ich led to the federal gov- ernment regul ating their use and manufacture . Pest icide manu facturers have a variety of physical / · and chemical disposal methods for pest icide wast es. Dis- posal or detoxification me thods for pesticides that have become enviro nmental pollutants through sp ills or accumu- la tion in · high use areas are limited or nonexistent . Th ere 1s a �otential for biodegradation to help decontaminate these environmental pollutants, since the degradation of organic compounds in the environment is predominantly micro- biologic ally mediated . Further research on microbial metabolism of xenobioti�s and their ability to degrade these comp ound s under different growth condit ions is needed. 2 Four objectives for this thesis research were to: 1. Screen lab oratory field isolates for their· capability to degrade the pesticides alachl or, carbof uran, and dicamba. 2a . Take key isolates obtained from objective 1 and determine in vitro broth degradat ion in three types of culture media. 2b . With these same isolates determine thei� in vitro soil slurry degradation capability . 3. Determine alachlor degradation by eight isolates obt ained from an agricultural chemical spill. 4. Determine if any observed pest icide degradation was mediated . by plasmid or chromoso mal DNA . 3 CHAPTER 1 LITERATURE REV IEW Pesticide degradat ion occurs thr ough chemical and _ biological metabolic processes wh ich are char acteri zed as either abiotic or biotic, respectively (8, 48). Abiotic degradat ion is not dependent on specific soil enzymes, and involves chemical reactions with other organic mole cule s. A me tabolic reaction is one wh ere alterat ions in the pesti­ cide are mediated by an enzyme . ABIOTIC DEGRADATION EFFECTS MED IATED BY HUMUS FORMA TION: Ab iotic degradation of pesticides may occur · during humus form ation in the soil. The organic fraction of soil may contain carboxyl, phenolic, aliphatic , hydroxyl, carbon yl, as well as le ss frequently found functional groups such as amino, imino , and sulfhydryl groups (1, 8). These highly react ive functional groups link together chemically with polysacchar­ ides , am ino acids , peptides and proteins , and other organic matter. This results iri a complex �ixture of polymerized material called humus and pesticid� molecule s or their me t abol ites may become incorporated during " its form ation ( 8 ) . 4 Mic roorganisms may produce enzymes that gen erate highly reactive pesticid e molecule s that are otherwis e unreactive . Th ese then combine with functional groups present in the soil organic ma tter. For example phenoli c pesticides can be transf ormed in to active phenolic radic als by the action of phenoloxidases (8) . Th ese radicals then stabil ize by linkin g together or by further oxid ation to quinones. EFFECTS MED I ATED BY. CHANGE OF pH : The hydrogen ion concentration has a maj or effect on the reactivit y of the pest icid� mo lecule by ionizing func tional groups of pesticid es or functio nal groups present in soil organic mat ter (8, 64 ).