the effect(s) of humic substances on motility, chemotaxis and degradation of...
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![Page 1: THE EFFECT(S) OF HUMIC SUBSTANCES ON MOTILITY, CHEMOTAXIS AND DEGRADATION OF 2,4-DICHLOROPHENOXYACETIC ACID DEGRADING SOIL BACTERIA Ted I. Brewster Department](https://reader031.vdocuments.net/reader031/viewer/2022020117/56649ceb5503460f949b6248/html5/thumbnails/1.jpg)
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
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Biological Remediation of Biological Remediation of Polluted Soils Using Humic-Polluted Soils Using Humic-
Mediated TechnologyMediated Technology
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Oil Field Drilling Site in Western China
Tank Bottom Fines in Texas Oil Field Close up of Tank Bottom Fines
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How do Humic How do Humic Substances Effect Substances Effect the Degradation of the Degradation of
2,4-D?2,4-D?
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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.
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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?
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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
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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
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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
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Is the Is the Microorganism Microorganism
Motile?Motile?
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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
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Is the Is the Microorganism Microorganism Chemotactic?Chemotactic?
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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
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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
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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
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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
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Growth Curve Analysis Growth Curve Analysis ResultsResults
Ralstonia eutrophiaRalstonia eutrophia JMP JMP 134134
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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
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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
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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
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Additional Analysis (Drop Additional Analysis (Drop Assay)Assay)
Drop Assay with or
w/o ring of turbidity
Gel Scanner
Recorder
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Drop Analysis ResultsDrop Analysis ResultsRepresentative Representative Photographs of Photographs of Ring of Ring of TurbidityTurbidity
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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
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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
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Drop Assay Results Drop Assay Results ComparisonComparison
Bugs Only Bugs & Leonardite
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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
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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.
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