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)
Methods
Sacrifice Worm
and Sterilize
Exterior
Earthworm Bacteria
Removal
-Whole Worm and Gut
Bacteria
Cultured on
Various Media
-Aerobic and Anaerobic
Samples Are Taken
at Time Points to
Monitor Ergovaline
Degradation
Ergovaline is
extracted by
Chloroform
Cultures
Inoculated with
Ergovaline
Samples Are
Purified Using a
Solid Phase
Extraction
Syringe
Cultures BackTransferred Twice
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
Internal
Standard
Black – 0 hrs
Blue – 24 hrs
Red – 48 hrs
Green – 72 hrs
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?
E-mail:
Durhamn@onid.orst.edu