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

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 10 4 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 10 9 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 2-Chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5- s -triazine The Herbicide that Launched a Thousand Careers 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!

Atrazine • 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 27 o C. • Half life in soil: 4 to 57 weeks. • Frequent source of crop damage and ground water contamination.

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

Atrazine can Serve as Sole Nitrogen Source * 3*CO 2 + 5 NH 3 * *

Enrichment Cultures using Atrazine as Sole N Source for Growth Soil from Minnesota Spill Site ~ 14,000 ppm atrazine Wash Soil and Centrifuge Defined Medium N – Atrazine Buffer C – citrate + sucrose Soil Subculture every 2 weeks, check atrazine degradation Consortium of Degraders Transfer to Solid Medium Pure Successive Culture Atrazine + Nutrient restreaking Agar

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 m m

Substrate Range of Atrazine Chlorohydrolase (AtzA) from Pseudomonas ADP Degraded Not Degraded Atrazine Deethyldeisopropyl- atrazine Simazine Melamine Deisopropylatrazine Terbuthylazine

Strains Tested for Sequence Homology to atzABC Strain Genus State Year Isolated 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 Location of Strain 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 Nebraska 99.1 100 100 J14a 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 OriV Apa I atzE atzF Not I atzD tra operon Xho I 80-100% 99% identity DNA sequence to pR751 identity to pR751 trf A 100000 trb operon 80-100% 20000 pADP-1 DNA sequence Nru I identity to pR751 80000 99% identity tnp A 108,845 bps to pR751 Eco RV 40000 IS 1071 IS 1071 tnpA 60000 atzA Xba I tnpA atz C IS 1071 Sac I atz B tnpA Mer IS 1071 II Pvu Operon

Complete Pathway for Atrazine Degradation by Pseudomonas sp. Strain

Evolution of Bacterial s -Triazine Hydrolases Was there a common ancestor?

98% Identity with Different Cl Functionality! N N N N N Atrazine HO N N N N N H 2 N N N N N N Melamine

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

Arthrobacter aurescens TC1 Plasmid-encoded O R H - R C N C CO 2 H R - H 2 N C CO 2 RH 2 CC N H C N Amino Phenylacetate - CO 2 acid N permease permease R 1 O R O RH 2 CC NH 2 - N N H 2 N C CO 2 CNH 2 NH 2 H R 3 R 2 R N N N O H H - O CO 2 N - - RCH 2 CO CO 2 s -triazines NH 2 NH 2 C O NH N N -O 2 C NH H - CO 2 + -O 2 C NH 2 H 2 N H 2 NCH 2 R NH 3 + O -O 2 C NH 3 H 2 N NH 2 O + NH 3 H HC R O N NH 2 -O 2 C - N C S CO 2 H CH 3 N NH 2 O -O 2 C N EPTC H 2 N N H H HO N (herbicide) Spermine O OH -2 -O 2 C N PO 3 CNHCH 3 H O H 3 C Glyphosate O - N HO N H 3 C (herbicide) O OH OH Carbaryl H N HO N OH (insecticide) CH 3 Cl O HO H 3 C Cl (-)- Synephrine N N H Nicotine H 3 C (bitter orange) Diuron (natural insecticide) N OH (herbicide) 2-Hydroxypyridine Arthrobacter species Plasmid-encoded

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 NO 3 or NH 4 .

Technology Applications Remediation of s-Triazines in the Environment  Soil Remediation  Water Remediation

Soil Remediation – GEM s Atrazine Spill Site surface 4,600 Spill = 250 gal tank of field-ready atrazine 35 yd 3 of soil isolated Time = 18 months 1 ft 29,000 • Initial spill: [Atrazine] = 11,500 ppm 3,600 400 1,400 (uniform distribution) 2.5 ft 2,000 500 3,000 700 800 600 • Regulatory limit = 2ppm field 3,000 700 2,500 application, 3ppb drinking water 4 ft 400 1,150 3,700 5 ft 3,300 3,600 • Indigenous microbes: ~70% reduction 2,400 in 18 months ( half-life = 350 days ) : diameter = 20 feet ground level • Applied enzyme: ~77% reduction in 8 weeks ( half-life = 31 days ) LANDFARMED JUNE 2000 - Over 80+ acres of field sorghum

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 Medicago sativa 5 ppm Atrazine 5 ppm Atrazine Transgenic Wild-Type Transgenic Wild-Type

Grass Plants Transformed with p-atzA Tall Fescue ( Festuca arundinacea ) • Perennial ryegrass ( Lolium perenne ) • Switchgrass ( Panicum virgatum ) • Alfalfa ( Medicago sativa ) •