Earthworms Possible Plant Detoxification and Bioremediation Tool Nick Durham Dr. A.M. Craig (Mentor) College of Veterinary Medicine

Willamette Valley Grass Seed Grass seed is one of Oregon’s largest and most important agricultural products [$300 million] Tall fescue is the most highly harvested grass The seed is first harvested and then the remaining plant material is sold as straw[$65 million]

The Endophyte 95% of tall fescue is infected with the endophyte Neotyphodium coenophialum This endophyte beneficially provides the plant with –Drought resistance –Resistance to insects Negatively, this endophyte produces ergot alkaloids which are toxins

Ergot Alkaloids Toxic to livestock that feed on straw hay Cause vasoconstriction –Livestock cannot regulate temperature Responsible for $1 billion loss to livestock producers each year in the United States

Ergovaline and Ergotamine Two of the ergot alkaloids found in tall fescue Nitro-aromatic compounds that are difficult to degrade

How Would Bacteria Help? External Solution: Degrade the toxins on straw before it is consumed Internal Solution: Degrade the toxins inside the livestock before they have a chance to go systemic and cause deleterious effects

Background: Why Earthworms? Earthworms can survive in toxic environments that contain ergot alkaloids Earthworms have bacteria that produce nitrogen gas Earthworms contain bacteria capable of reducing nitrogen compounds and degrading nitro aromatic compounds

Background: The Industry A group of grass seed farmers in Halsey, OR have been composting grass seed with earthworms.

Earthworms are living on endophyte infected grass seed in a compost pile. Earthworms have been thriving in an extremely toxic environment

Link How can the earthworms survive such a toxic environment? Can the bacteria in earthworms degrade toxic nitro-aromatic compounds? Could these bacteria be used to protect cattle for toxicosis?

Objective Characterize a method for monitoring the degradation of ergovaline by earthworms Characterize the ability of earthworm bacteria to degrade ergovaline

Hypothesis The bacteria from earthworms will be able to degrade Ergovaline into nontoxic intermediates This degradation can be monitored by HPLC (High Performance Liquid Chromatography).

Making Compost We made two compost bins We added a great deal of tall fescue seed which contains the highest level of toxins We spiked each bin with a different type of earthworm Red Wiggler(Eisenia foetida) Nightcrawler(Lumbricus rubellus)

Worms Thrived (Red Wigglers)

Sacrifice Worm and Sterilize Exterior Earthworm Bacteria Removal -Whole Worm and Gut -Aerobic and Anaerobic Bacteria Cultured on Various Media Cultures Back- Transferred Twice Cultures Inoculated with Ergovaline Samples Are Taken at Time Points to Monitor Ergovaline Degradation Methods Ergovaline is extracted by Chloroform Samples Are Purified Using a Solid Phase Extraction Syringe Final Product is analyzed by HPLC

HPLC of Ergot Alkaloids

Media and Environment Rich anaerobic environment –Lots of nutrients, but no oxygen –Cometabolize toxin as they grow 6 anaerobic consortiums were grown and tested for the ability to degrade ergovaline. The level of ergovaline in the consortium was monitored at 24 hour intervals for 72 hours

Consortium 1 - HPLC Black – 0 hrs Blue – 24 hrs Red – 48 hrs Green – 72 hrs Internal Standard EV

Rich Anaerobic Trials All 6 consortiums of bacteria failed to degrade any ergovaline I did not find the right consortium Maybe there was too much food, too many choices

Targeting Ergovaline Peptide Bond Tripeptide: Proline, Valine, and Alanine Potential Peptide Target

Media and Environment Whole protein diet of milk casein –Lots of peptide bonds No free amino acids –Grown with and without oxygen 12 consortiums (6 aerobic/6 anaerobic) were grown and tested for the ability to degrade ergovaline. The level of ergovaline in the consortium was monitored for 24 hours

Casein Trials All 12 consortiums of bacteria failed to degrade any ergovaline I did not find the right consortium Maybe the bacteria still had casein left so it did not need to eat the ergovaline

Currently Two new approaches –Minimal media: Force bacteria to learn to eat everything –Concentrate cells: Increase cell density while decreasing concentration of casein and add ergovaline

Summary of Objectives I have successfully characterized a method for monitoring the degradation of ergovaline by earthworms As of yet, none of my cultured bacteria have been capable of degrading ergovaline

Acknowledgements Dr. A. Morrie Craig Nevon Dernstein Department of Microbiology Howard Hughes Medical Institute Dr. Kevin Ahern

Questions?