The Power of Soil! The Breakdown

of 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

on 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

NN

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

of 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

CO2-

NH2

CNH

NH

NHO

-O2CCO2

-

NH3+

-O2C NH

PO3-2

HN

CH3

OH

HO

N OH

Cl

ClNH

NH3C

H3C

O

N

N

N

NH

NH

R1

R3 R2

N

C N

NH2

O

-O2C NH

H2NNH2

+

-O2C NH

CH3

N OHHO

OH

H2N NH2

O-O2C

NH3+

-O2C

HN NH2

O

H3CN O-

H3C

O

N C

O

S

OCNHCH3

O

H2N NH

HN NH2

N

OH

HO

N

CO2-

HO

H2NCH2R

NH3

R CHN C

H

O

CO2-

R

H2N CH

CO2-

R

RH2CC NH2

O

RCH2CO2-

H2N CH

CO2-

R

N

CO2-

N

CNH2

O

R

CO2-

O

HC R

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

RH2CC N

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 occur 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?