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 Tank Bottom Fines in Texas Oil Field Close up of Tank Bottom Fines

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

6. 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.

7. 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?

8. Soil Microorganism Isolation Sample Soil (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 on MMO/2,4-DIsolate ColoniesStreak Isolates Grow Isolates in Liquid Media and on Agar Plates (MMO/2,4-D) Grow in liquid media and on agar plates (PTGY) Continually transfer isolates every 24 hours in both MMO/2,4-D and PTGY media’s

9. 2,4-D Degradation & Growth Analysis Simple growth in MMO liquid media with 0.05% 2,4-D Growth Curve experiment

10. Growth Curve Experiment 100 ml MMO/w 2,4-D (0.05%) Shake1 ml sampleSpin Supernant Pellet 1 ml MMO Analyze @ 296.7 nm Analyze @ 595 nm UV/IR Scanner 1 ml Culture + + Every 1-1.5 hour

11. Is the Microorganism Motile?

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

13. Is the Microorganism Chemotactic?

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

15. The Effect(s) of Humic Substances on 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

16. Growth Curve Experiment Using Ralstonia eutrophia JMP 134 and Soil Isolate Shake1 ml sample Pellet 1 ml MMO Analyze @ 296.7 nm Analyze @ 595 nm UV/IR Scanner 1 ml Culture + + Every 1-1.5 hour 100 ml MMO/2,4-D (0.05%) + 125 ul Leonardite Spin

17. 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

18. Growth Curve Analysis Results Ralstonia eutrophia JMP 134

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

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

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

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

23. Drop Analysis Results Representative Photographs of Ring of Turbidity

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

25. Drop Assay Results (Isolate/2,4-D/Leonardite) Representative Graphs from Gel Scanner Initial Scan 0 Hours3.5 Hours 24 Hours 1 Hour

26. Drop Assay Results Comparison Bugs Only Bugs & Leonardite

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

28. Conclusion/Discussion Humic substances do not inhibit the degradation of 2,4-D by R. eutrophia and the soil isolate. 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 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. Humic Substances do not inhibit the chemotaxtis or motility of the soil isolate.