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Ted I. Brewster Department of Microbiology Brigham Young University

THE EFFECT(S) OF HUMIC SUBSTANCES ON MOTILITY, CHEMOTAXIS AND DEGRADATION OF 2,4-DICHLOROPHENOXYACETIC ACID DEGRADING SOIL BACTERIA. Ted I. Brewster Department of Microbiology Brigham Young University. Biological Remediation of Polluted Soils Using Humic-Mediated Technology.

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Ted I. Brewster Department of Microbiology Brigham Young University

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  1. THE EFFECT(S) OF HUMIC SUBSTANCES ON MOTILITY, CHEMOTAXIS AND DEGRADATION OF 2,4-DICHLOROPHENOXYACETIC ACID DEGRADING SOIL BACTERIA Ted I. Brewster Department of Microbiology Brigham Young University

  2. Biological Remediation of Polluted Soils Using Humic-Mediated Technology

  3. Oil Field Drilling Site in Western China Close up of Tank Bottom Fines Tank Bottom Fines in Texas Oil Field

  4. How do Humic Substances Effect the Degradation of 2,4-D?

  5. Analyses Performed • Soil Microorganism Isolation Procedures • Growth on 2,4-D • Growth Curve Analyses (2,4-D degradation vs. Microorganism growth) • Swarm Plate Analyses ( Motility) • Drop Plate Analyses (Chemotaxis) • Soil Microorganism Characteristics Gram Stain, Growth on Mercury Plates, API NFT, etc.

  6. Isolating Microorganism(s) for Proposed Study • Does the microorganism grow on 2,4-D? • Does the microorganism grow quickly? • Is the Microorganism motile? • Is the Microorganism Chemotactic towards 2,4-D?

  7. Soil Microorganism Isolation SampleSoil (5 g) Add to H2O (95 ml) Shake (24-72 hours) Sample (1 ml) Add to 100 ml MMO w/2,4-D (0.05%) Streak Isolates Isolate Colonies Streak on MMO/2,4-D Continually transfer isolates every 24 hours in both MMO/2,4-D and PTGY media’s Grow Isolates in Liquid Media and on Agar Plates (MMO/2,4-D) Grow in liquid media and on agar plates (PTGY)

  8. 2,4-D Degradation & Growth Analysis • Simple growth in MMO liquid media with 0.05% 2,4-D • Growth Curve experiment

  9. Growth Curve Experiment Every 1-1.5 hour + 100 ml MMO/w 2,4-D (0.05%) 1 ml Culture Shake 1 ml sample Spin + 1 ml MMO Analyze @ 595 nm Pellet Analyze @ 296.7 nm UV/IR Scanner Supernant

  10. Is the Microorganism Motile?

  11. Motility (Swarm Plate Assay) Touch center of plate streak Liquid MMO/2,4-D (0.05%) PTGY Agar Plate Toothpick Sample Swarm Plate Look for growth to cover entire plate

  12. Is the Microorganism Chemotactic?

  13. Chemotaxis (Drop Plate Assay) 1 ml Culture Transfer Discard Supernant Spin 40 ml MMO/2,4-D Shake (18-24 hours) Harvest Pour into 60 mm Petri Dish Add 12 ml Buffer & 3 ml Hydroxy. . . To Pellet Spin Resuspend Pellet w/25 ml PBS Repeat 3 x’s Discard Supernant Look for Ring of Turbidity to form in direct relation w/2,4-D concentration as it diffuses into medium Add 2,4-D Crystal to center of plate

  14. The Effect(s) of Humic Substanceson the Degradation & Chemotaxis of 2,4-D • Growth Curve Experiment Using Ralstonia eutrophia JMP 134 and Soil Isolate Using Leonardite as the humic substance • Swarm Plate Experiment using Soil Isolate Using Leonardite, Soil Fulvic Acid, Peat Fulvic Acid, Soil Humic Acid, and Peat Humic Acid • Drop Plate Assay Using Soil Isolate with 2,4-D Using Leonardite, Soil Fulvic Acid, Peat Fulvic Acid, Soil Humic Acid, and Peat Humic Acid

  15. Growth Curve Experiment Using Ralstonia eutrophia JMP 134 and Soil Isolate Every 1-1.5 hour + 100 ml MMO/2,4-D (0.05%) + 125 ul Leonardite 1 ml Culture Shake 1 ml sample Spin + 1 ml MMO Analyze @ 595 nm Pellet Analyze @ 296.7 nm UV/IR Scanner

  16. Test Samples • Controls (in Triplicate) • No Soil Isolate and no Leonardite • Soil Isolate with Leonardite and no 2,4-D • Soil Isolate with 2,4-D • Samples (in Triplicate) • Soil Isolate with 125 ul Leonardite

  17. Growth Curve Analysis ResultsRalstonia eutrophia JMP 134

  18. Swarm Plate Experiment Touch center of plate streak Liquid MMO/2,4-D (0.05%) PTGY Agar Plate Toothpick Sample Swarm Plate w/humic substance Each swarm plate was prepared with 125 ul humic substances: Leonardite, Soil Fulvic Acid, Peat Fulvic Acid, Soil Humic Acid, and Peat Humic Acid Look for growth to cover entire plate

  19. Swarm Plate Analysis Results • Swarming activity was not affected by the presence of the humic substances

  20. Drop Plate Assay Analysis 1 ml Culture Transfer Discard Supernant Spin 40 ml MMO/2,4-D Shake (18-24 hours) Harvest Add 25 ml PBS Pour into 60 mm Petri Dish Add 12 ml Buffer & 3 ml Hydroxy. . . and 100 ul humic substance To Pellet Spin Pellet Repeat 3 x’s Discard Supernant Look for Ring of Turbidity to form in direct relation w/2,4-D concentration as it diffuses into medium Add 2,4-D Crystal to center of plate

  21. Additional Analysis (Drop Assay) Drop Assay with or w/o ring of turbidity Recorder Gel Scanner

  22. Drop Analysis Results • Representative Photographs of Ring of Turbidity

  23. Drop Assay Results (Isolate/2,4-D Only) • Representative Graphs from Gel Scanner 3.5 Hours Initial Scan 0 Hours 24 Hours

  24. Drop Assay Results (Isolate/2,4-D/Leonardite) • Representative Graphs from Gel Scanner Initial Scan 24 Hours 1 Hour 0 Hours 3.5 Hours

  25. Drop Assay Results Comparison Bugs & Leonardite Bugs Only

  26. Drop Assay Results (Other Humic Substances) Soil Humic 3.5 Hours Soil Fulvic 3.5 Hours Peat Fulvic 3.5 Hours Peat Humic 3.5 Hours

  27. Conclusion/Discussion • Humic substances do not inhibit the degradation of 2,4-D by R. eutrophia and the soil isolate. • Humic Substances may assist the soil microorganisms in their ability to degrade 2,4-D • Humic Substances do not inhibit the chemotaxtis or motility of the soil isolate.

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