THE EFFECT(S) OF HUMIC THE EFFECT(S) OF HUMIC SUBSTANCES ON MOTILITY, SUBSTANCES ON MOTILITY,

CHEMOTAXIS AND CHEMOTAXIS AND DEGRADATION OF 2,4-DEGRADATION OF 2,4-

DICHLOROPHENOXYACETIC DICHLOROPHENOXYACETIC ACID DEGRADING SOIL ACID DEGRADING SOIL

BACTERIABACTERIA

Ted I. BrewsterTed I. Brewster

Department of MicrobiologyDepartment of Microbiology

Brigham Young UniversityBrigham Young University

Biological Remediation of Biological Remediation of Polluted Soils Using Humic-Polluted Soils Using Humic-

Mediated TechnologyMediated Technology

Oil Field Drilling Site in Western China

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

How do Humic How do Humic Substances Effect Substances Effect the Degradation of the Degradation of

2,4-D?2,4-D?

Analyses PerformedAnalyses Performed

Soil Microorganism Isolation ProceduresSoil Microorganism Isolation Procedures

Growth on 2,4-DGrowth on 2,4-D

Growth Curve Analyses (2,4-D Growth Curve Analyses (2,4-D degradation vs. Microorganism growth)degradation vs. Microorganism growth)

Swarm Plate Analyses ( Motility)Swarm Plate Analyses ( Motility)

Drop Plate Analyses (Chemotaxis)Drop Plate Analyses (Chemotaxis)

Soil Microorganism CharacteristicsSoil Microorganism CharacteristicsGram Stain, Growth on Mercury Plates, API NFT, Gram Stain, Growth on Mercury Plates, API NFT, etc.etc.

Isolating Microorganism(s) Isolating Microorganism(s) for Proposed Studyfor Proposed Study

Does the microorganism grow on 2,4-D?Does the microorganism grow on 2,4-D?

Does the microorganism grow quickly?Does the microorganism grow quickly?

Is the Microorganism motile?Is the Microorganism motile?

Is the Microorganism Chemotactic Is the Microorganism Chemotactic towards 2,4-D? towards 2,4-D?

Soil Microorganism Soil Microorganism IsolationIsolation

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

2,4-D Degradation & Growth 2,4-D Degradation & Growth AnalysisAnalysis

Simple growth in MMO liquid media Simple growth in MMO liquid media with 0.05% 2,4-Dwith 0.05% 2,4-D

Growth Curve experimentGrowth Curve experiment

Growth Curve Growth Curve ExperimentExperiment

100 ml MMO/w 2,4-D (0.05%)

Shake 1 ml sample Spin

Supernant

Pellet

1 ml

MMO

Analyze @ 296.7 nm

Analyze @ 595 nm

UV/IR Scanner

1 ml Culture

+

+

Every 1-1.5 hour

Is the Is the Microorganism Microorganism

Motile?Motile?

Motility (Swarm Plate Motility (Swarm Plate Assay)Assay)

Liquid MMO/2,4-D (0.05%)

streak

PTGY Agar Plate Toothpick Sample Swarm Plate

Touch center of plate

Look for growth to cover entire plate

Is the Is the Microorganism Microorganism Chemotactic?Chemotactic?

Chemotaxis (Drop Plate Chemotaxis (Drop Plate Assay)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

The Effect(s) of Humic SubstancesThe Effect(s) of Humic Substanceson the Degradation & Chemotaxis on the Degradation & Chemotaxis

of 2,4-Dof 2,4-DGrowth Curve Experiment Using Growth Curve Experiment Using Ralstonia Ralstonia eutrophiaeutrophia JMP 134 and Soil Isolate JMP 134 and Soil Isolate

Using Leonardite as the humic substanceUsing Leonardite as the humic substance

Swarm Plate Experiment using Soil IsolateSwarm Plate Experiment using Soil IsolateUsing Leonardite, Soil Fulvic Acid, Peat Fulvic Using Leonardite, Soil Fulvic Acid, Peat Fulvic Acid, Soil Humic Acid, and Peat Humic AcidAcid, Soil Humic Acid, and Peat Humic Acid

Drop Plate Assay Using Soil Isolate with Drop Plate Assay Using Soil Isolate with 2,4-D2,4-DUsing Leonardite, Soil Fulvic Acid, Peat Fulvic Using Leonardite, Soil Fulvic Acid, Peat Fulvic Acid, Soil Humic Acid, and Peat Humic AcidAcid, Soil Humic Acid, and Peat Humic Acid

Growth Curve Experiment Using Growth Curve Experiment Using Ralstonia eutrophiaRalstonia eutrophia JMP 134 and JMP 134 and

Soil IsolateSoil Isolate

Shake 1 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

Test SamplesTest Samples

Controls (in Triplicate)Controls (in Triplicate)No Soil Isolate and no LeonarditeNo Soil Isolate and no Leonardite

Soil Isolate with Leonardite and no Soil Isolate with Leonardite and no 2,4-D2,4-D

Soil Isolate with 2,4-DSoil Isolate with 2,4-D

Samples (in Triplicate)Samples (in Triplicate)Soil Isolate with 125 ul LeonarditeSoil Isolate with 125 ul Leonardite

Growth Curve Analysis Growth Curve Analysis ResultsResults

Ralstonia eutrophiaRalstonia eutrophia JMP JMP 134134

Swarm Plate ExperimentSwarm Plate Experiment

Liquid MMO/2,4-D (0.05%)

streak

PTGY Agar Plate Toothpick Sample Swarm 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

Swarm Plate Analysis Swarm Plate Analysis ResultsResults

Swarming activity was not affected Swarming activity was not affected by the presence of the humic by the presence of the humic substancessubstances

Drop Plate Assay Drop Plate Assay AnalysisAnalysis

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-D Harvest

Pellet

Add 25 ml PBS

Additional Analysis (Drop Additional Analysis (Drop Assay)Assay)

Drop Assay with or

w/o ring of turbidity

Gel Scanner

Recorder

Drop Analysis ResultsDrop Analysis ResultsRepresentative Representative Photographs of Photographs of Ring of Ring of TurbidityTurbidity

Drop Assay Results Drop Assay Results (Isolate/2,4-D Only)(Isolate/2,4-D Only)

Representative Graphs from Gel Representative Graphs from Gel ScannerScanner

Initial Scan0 Hours

3.5 Hours

24 Hours

Drop Assay Results Drop Assay Results (Isolate/2,4-D/Leonardite)(Isolate/2,4-D/Leonardite)

Representative Graphs from Gel ScannerRepresentative Graphs from Gel Scanner

Initial Scan

0 Hours 3.5 Hours

24 Hours

1 Hour

Drop Assay Results Drop Assay Results ComparisonComparison

Bugs Only Bugs & Leonardite

Drop Assay Results (Other Humic Drop Assay Results (Other Humic Substances)Substances)

Peat Fulvic 3.5 Hours

Peat Humic 3.5 Hours

Soil Fulvic 3.5 Hours

Soil Humic 3.5 Hours

Conclusion/DiscussionConclusion/Discussion

Humic substances do not inhibit Humic substances do not inhibit the degradation of 2,4-D by the degradation of 2,4-D by R. R. eutrophiaeutrophia and the soil isolate. and the soil isolate.

Humic Substances may assist the Humic Substances may assist the soil microorganisms in their soil microorganisms in their ability to degrade 2,4-Dability to degrade 2,4-D

Humic Substances do not inhibit Humic Substances do not inhibit the chemotaxtis or motility of the chemotaxtis or motility of the soil isolate.the soil isolate.