The Power of Soil! The Breakdown of Pollutants by Soil - - PowerPoint PPT Presentation

The Power of Soil! The Breakdown

• f Pollutants by Soil

Microorganisms

Michael J. Sadowsky

Department of Soil, Water, and Climate; and Biotechnology Institute University of Minnesota, St. Paul, MN (USA)

Soil as a Habitat

 Texture/structure  Charged sites  Soil microbial activity

 Factors that influence soil microbial

activity

• Temperature
• pH
• Air/water
• Pesticides/pollutants
• Rhizosphere effect

Many microbial processes are affected by soil water/oxygen availability

Temperature

Pesticides/pollutants can decrease microbial survival

Soil as a Habitat

5,000 – 10,000 species/gram soil Each species about 104 cells/g

Rhizosphere

 Area immediately surrounding plant

roots

 Much higher concentrations of

microorganisms than the bulk soil

 Interaction between plants and

inhabitants of rhizosphere

 May be up to 109 cells/g in rhizosphere

Distribution of microorganisms in the soil Microbial numbers decline with depth because of declining nutrient availability Major discrepancy between viable and total counts

Antoni van Leeuwenhoek

 1670s Antoni

van Leeuwenhoek discovered bacteria and protozoa (“animalcules”)

How are organisms classified?

 Historically – structure, morphology,

staining reactions, physiological abilities

 Today – molecular methods show

relationships among organisms

The universal phylogenetic tree (Bull and Wichman, 2001)

Roles of bacteria in nature

 Decomposition  Nutrient cycling  Symbionts  Pathogens  Bioremediation  Biocontrol

Microbial Catabolic Enzymes Transform

• Natural products
• Synthetic compounds produced via human

activity

• Compounds derived from abiotic reactions

Xenobiotics

 chemicals synthesized by humans that

have no close natural counterparts (e.g. plastics and pesticides)

 Literally means – stranger to organisms

Many of these are also chlorinated compounds that are recalcitrant to biodegradation, such as :

Industrial Solvents Propellants Flame Retardants Pesticides and Herbicides

Atrazine

The Herbicide that Launched a Thousand Careers

2-Chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-s-triazine

The most widely used s-triazine herbicide in the United States.

Intensive Inputs of Chemicals into the Environment Place Tremendous Selection Pressure on Microorganisms

Over 136 Million Pounds of s-triazine herbicides are used in the US per year!

• Developed in early 1950’s by Geigy Co.
• Used as a pre/post emergent herbicide or

total herbicide for primarily corn.

• Water solubility: 33 mg/L (33 ppm) at 27oC.
• Half life in soil: 4 to 57 weeks.
• Frequent source of crop damage and ground

water contamination.

Atrazine

Brief History of Atrazine Biodegradation

Before 1993

• Atrazine biodegradation shown in mixed microbial

cultures

• Many s-triazine-degrading bacteria isolated, none

mineralize atrazine

After 1993

• Numerous pure atrazine-degrading bacteria isolated
• Molecular Basis of atrazine biodegradation started to

be revealed

3*CO2 + 5 NH3

* * *

Atrazine can Serve as Sole Nitrogen Source

Enrichment Cultures using Atrazine as Sole N Source for Growth

Soil from Minnesota Spill Site ~ 14,000 ppm atrazine

Soil Buffer

Wash Soil and Centrifuge

Defined Medium N – Atrazine C – citrate + sucrose Subculture every 2 weeks, check atrazine degradation

Consortium of Degraders

Transfer to Solid Medium

Atrazine + Nutrient Agar

Successive restreaking

Pure Culture

Pseudomonas sp. Strain ADP

• Degrades atrazine to

carbon dioxide and ammonia.

• Uses atrazine as a sole

source of nitrogen.

• Atrazine degradation

phenotype can be distinguish by the formation of clear zones

• n media containing

atrazine.

2 mm

Substrate Range of Atrazine Chlorohydrolase (AtzA) from Pseudomonas ADP

Degraded Atrazine Simazine Deisopropylatrazine Terbuthylazine Not Degraded Deethyldeisopropyl- atrazine Melamine

Strains Tested for Sequence Homology to atzABC

Strain Genus State Isolated Year Reported

ADP Pseudomonas MN 1995 M91-3 Ralstonia OH 1995 ATZ1 Clavibacter CA 1996 38/38 Unknown IN 1996 SG1 Alcaligenes LA 1998

Atrazine-Degrading Bacteria with atz Genes

% DNA Sequence Identity Strain Location of Isolation

atzA atzB atzC Pseudomonas ADP Minnesota 100 100 100 Alcaligenes SG1 California 99.2 100 100 Ralstonia M91-3 Ohio 99 100 100 Agrobacterium J14a Nebraska 99.1 100 100 Isolate 38/38 Indiana 99.3 100 99.8

Bacterial genera currently reported to transform s-triazine compounds

Pseudomonas ADP Agrobacterium tumefaciens Rhodococcus rhodochrous Sphingomonas yaniokuyae Streptomyces strain PS1/5 Flavobacterium oryzihabitans Acinetobacter calcoaceticus Variovorax paradoxus Chelatobacter heintzii Arthrobacter aurescens TC1 Aminobacter aminovorans Chelatobacter heintzii Cit1 Stenotrophomonas maltophilia Bacillus sp. RK016 Pseudaminobacter sp. C223, C147, C195 Stenotrophomonas maltophilia Nocardioides sp. C190 Delftia acidovorans D24 Clavibacter michiganese Exiguobacterium sp. BTAH1 Agrobacterium radiobacter Bacillus licheniformis Bacillus megaterium

Atrazine Catabolic Plasmid, pADP-1

pADP-1

108,845 bps

20000 40000 60000 80000 100000

Not I Nru I Eco RV Pvu II I Sac I Xba I Xho OriV tra operon tnpA IS1071 atzA tnpA IS1071 atzB tnpA IS1071 Mer atzC tnpA IS1071 trb operon trf A 99% identity to pR751 99% identity to pR751 Apa I

atzD atzE atzF

80-100% DNA sequence identity to pR751 80-100% DNA sequence identity to pR751 Operon

Complete Pathway for Atrazine Degradation by Pseudomonas sp. Strain

Evolution of Bacterial s-Triazine Hydrolases

Was there a common ancestor?

N N N Cl N N N N N H2N N N N N N HO N N

Atrazine Melamine

98% Identity with Different Functionality!

Where did atrazine chlorohydrolase (and TriA) come from?

Likely from another member of the Amidohydrolase Superfamily

• Cytosine deaminase and AtzA are the only known members
• f the amidohydrolase superfamily that contain Fe(II) as the

catalytic metal

Complete Genomic Sequencing of Arthrobacter aurescens TC1

 First Complete Genome of an Arthrobacter

• sp. strain

Arthrobacter Sequencing Project

 Funded by NSF  Contains a 4.8 Mbp Genome, 2

plasmids

 Sequenced by TIGR  Manually Annotated at UM

• NH2

• O2C

• NH3

• O2C

• 2

• O2C

• O2C

• O2C

• O2C

• HO

• R

• R

• H2N

• R

• N

• O

Amino acid permease Phenylacetate permease s-triazines EPTC (herbicide) Carbaryl (insecticide) Diuron (herbicide) Nicotine (natural insecticide) (-)- Synephrine (bitter orange) Glyphosate (herbicide) Spermine

Arthrobacter species Plasmid-encoded Arthrobacter aurescens TC1 Plasmid-encoded

2-Hydroxypyridine

Results of these studies indicate:

 Atrazine degradation ability has spread

to a large number of bacterial genera.

 Spread due to plasmid transfer and

transposition events.

 Nearly identical enzymes are involved

in atrazine mineralization.

Results, continued

 Evolution of atrazine degradation

ability happened relatively rapidly – 50 years!

 There are a few reported cases

where atrazine is losing efficacy due to proliferation of genes and bacteria, however this is likely not to

• ccur in soils containing sufficient

NO3 or NH4.

Technology Applications Remediation of s-Triazines in the Environment

 Soil Remediation  Water Remediation

Atrazine Spill Site

5 ft

Spill = 250 gal tank of field-ready atrazine 35 yd3 of soil isolated

• Initial spill: [Atrazine] = 11,500 ppm

(uniform distribution)

• Regulatory limit = 2ppm field

application, 3ppb drinking water

• Indigenous microbes: ~70% reduction

in 18 months (half-life = 350 days) :

• Applied enzyme: ~77% reduction in 8

weeks (half-life = 31 days) LANDFARMED JUNE 2000 - Over 80+ acres of field sorghum

4,600 29,000 800 3,600 2,400 3,000 3,000 2,000 500 3,700 1,150 400 ground level diameter = 20 feet 4 ft 2.5 ft

surface

1 ft 3,600 700 2,500 3,300 600 1,400 700 400 Time = 18 months

Soil Remediation – GEM s

Phytoremediation

Plant-based bioremediation strategies for the in situ treatment of contaminated soils, sediments, and groundwater

Phytoremediation - Transgenics

We have produced transgenic Medicago sativa, Nicotiana tabacum, and Arabidopsis plants containing bacterial atrazine chlorohydrolase (AtzA) to phytoremediate atrazine-contaminated soil and soil water

Nicotiana tabacum

Transgenic Wild-Type

Medicago sativa

Transgenic Wild-Type

5 ppm Atrazine 5 ppm Atrazine

Grass Plants Transformed with p-atzA

• Tall Fescue (Festuca arundinacea)
• Perennial ryegrass (Lolium perenne)
• Switchgrass (Panicum virgatum)
• Alfalfa (Medicago sativa)

Tall Fescue Hydroponics

0.5 µg/mL Atrazine 2.5 µg/mL Atrazine 6.5 µg/mL Atrazine 4.5 µg/mL Atrazine

WT TF-2008 WT TF-2008 WT TF-2008

WT TF-2008

Remediation of Drinking Water

Water Input

Bioreactor

AtzA AtzA AtzA AtzA AtzA AtzA AtzA AtzA

Silica Encapsulated Atrazine Degrading Bacteria

Long Scale Atrazine Degradation in Beads 4 months

Present and Future Goals

 Develop New Treatment Technologies

Using Genes, Enzymes, Plants, and Microbes

 Capitalize on Genomics-enabled Information

Collaborators

 Larry Wackett  Al Aksan



Jennifer Seffernick



Nir Shapir



Mervyn de Souza



Lisa Strong



Lin Wang



Charlotte Pedersen



Jeff Osborne



Gill Johnson



Betsey Martinez



Issac Fruchey



Jack Richmond



Many, many others

Funding From: USDA-NRI USGS Syngenta Inc.

• Univer. of Minnesota

BARD Consortium for Plant BioTech Research NSF

Thank you for inviting me and for your time and attention!

Questions?