Bioprospecting
By C Kohn, Waterford Agricultural Sciences
Some slides from “Bioprospecting for Cellulose-Degrading Microbes”, GLBRC
US Department of Energy Billion Ton
Challenge

The US DOE Billion Ton Challenge is to convert 1
billion tons of plant (cellulosic) biomass to ethanol
per year to replace 30% of current transportation
fuel use

Cellulose would be made into liquid fuel, create
electricity, and be used for energy-related chemical
reactions.
Biomass-cellulose and plant materials
(crops, grasses, trees, etc)
High energy/value products
• Liquid fuels (ethanol, biodiesel,
hydrocarbons, others)
• Energy sources (hydrogen, electricity)
• Chemical precursors
*Based on U.S. Energy
Consumption 2004
Renewable Fuels Energy Independence &
Security Act 2007 (EISA)

According to EISA, US production of corn-ethanol would
be capped at 15 billion gallons per year.

The additional energy needed would come from other sources,
particularly cellulosic energy.
40
Other
Biodiesel
Cellulosic
Corn
35
30
25
20
Biofuel
15
Other
Biodiesel
Cellulosic
Corn Ethanol
10
5
0
Year
Year:
2008
2010
2012
2014
2016
2018
2020
2007
4.7
2010
0.20
0.65
0.1
12
2022
4
1
16
15
Hurdles to Billion Ton and EISA

The biggest obstacle to these goals is the pretreatment
and hydrolysis step of producing cellulosic ethanol.
Hurdles to Billion Ton & EISA

While cellulose is the most abundant
biological material on the planet,
conversion of cellulose into ethanol is
currently made more difficult by the
challenge of breaking cellulose into
individual glucose molecules.

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Unless a feedstock can be converted into
glucose using enzymes or other chemicals
(such as strong acids), yeast cannot ferment
that feedstock into ethanol.
Better pre-treatment methods could make
cellulosic ethanol more available, more
sustainable, and less costly than gasoline.
Goal of pretreatment is to open up cell wall and
expose cellulose.
Hydrolysis with cellulase
Cellulose must be broken into individual glucose
molecules using cellulase enzymes before
fermentation can occur.
cellulose
enzyme
glucose
glucose
glucose
glucose
Improving Biomass Pretreatment

A key goal for improving pretreatment strategies is to discover
and improve natural cellulose-degrading enzymes


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To do this, we will need to find and extract them from diverse
environments
These environments may range from the highly toxic and
scalding environments of geysers to the deep layers of
tropical ant colonies to many other unique ecosystems
across the planet.
The search for these organisms is known as
bioprospecting.
Bioprospecting: the search for diverse organisms
for genes, biochemicals, and other compounds
that are of value to humans.
BIO-PROSPECTING

Bioprospecting can range
from trekking across the
Amazon to trekking across
the street to a cow pasture.
Genome Management Information System,
Oak Ridge National Laboratory
Http://genomics.energy.gov
Case-Study – Ant Agriculturalists


Fifty million years before humankind began farming, ancient
ants were already in the agriculture business.
Leaf-cutter ants have grown their own fungi crops long before
humans began their own agricultural production.

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Forager ants cut pieces of leaves and drag them to their nest.
Other ants chew the leaves to make a paste to feed their crop: fungi.
The ants have formed a mutualistic relationship with their fungus – in
exchange for feeding, growing, and protecting the fungus (and
protecting it from mold), ants can harvest part of the fungi for their
own food.
Specialized bacteria that live on the ants produce an antibiotic that
protects the fungi from the mold.
From Currie, C. 2001. Annual Reviews of Microbiology, Created by Cara
Gibson and adapted by Angie Fox
Leafcutter Ant
Symbiosis
Video
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PBS Video: Evolution:The Evolutionary Arms Race
View in: QuickTime | RealPlayer
YouTube
So why do we care?

So why do biofuel researchers care so much about ants,
fungus, antibiotics, and bacteria?

The reason this is critically important is because the ant
colony requires efficient plant degradation in order to
adequately feed the fungi that feeds the ants.

Research of these ant colonies has shown that a diverse
but consistent group of microbes enable an efficient
break-down of cellulose.

This provides a model for our own cellulose-degradation that
would be necessary for efficient, low-intensity biofuel
production.
Bioprospecting and Biofuels

Biofuel bioprospecting is really the search for enzymes
that already exist in yet-undiscovered living species.

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An enzyme is really just a protein that speeds up or slows
down a chemical reaction.
Enzymes have two key components:

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1. An Active Site.: This is where the substrate (the thing broken down,
e.g. cellulose) binds to the enzyme protein
2. A Regulatory Region: a region that
controls the activity of the enzyme
Each enzyme is specific to a
substrate
Substrates and Products

When a substrate (like cellulose) fits into the Active Site
of an enzyme, the Active Site binds to it and fits around it
(“Induced Fit”).

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This enables the enzyme to break apart the substrate
In the case of cellulose, it is broken down into individual
glucose molecules by the enzyme cellulase

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Glucose would be the product then in this reaction.
The products, e.g. glucose molecules,
leave the enzyme Active Site, and the
enzyme continues to break up more
cellulose.
Video: Click Here
Biochemical Pathways

The work of enzymes occurs in a specific, predictable
pattern and can be more than a one-step, one-enzyme
process.

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The process by which an enzyme breaks down a substrate is
called the biochemical pathway.
If multiple enzymes are involved, the product of the first
enzyme becomes the substrate of the
second enzyme.
This pathway continues until the final
product is made.
Enzymes in Biochemical Pathways
(click here for animation)
Bioprospecting

In the process of bioprospecting, researchers must
hypothesize where they might find species that produce
the enzymes that can most efficiently break down
cellulose.

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TPS: in what sorts of conditions or environments are we most
likely to find cellulose-degrading microbes?
Researchers must travel both short and great distances to
find, collect, isolate, and test these microbes.
Steps of Bioprospecting
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Once a microbe has been recovered…
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Step 1 - Plating: Spread the microbe on cellulose plates (agar
that contains only cellulose instead of other nutrients so that
only the cellulose consumers can survive)
Step 2 - Isolation: choose the fastest growing colonies.
Step 3 - Screening: test for cellulase activity
Step 4 – Community Method: incubate and test on different
feedstocks.
Summary

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While we have ambitious federal goals for the use of
biofuels to offset our use of fossil fuels, pretreatment and
hydrolysis of cellulosic feedstocks remains our largest
obstacle.
Bioprospecting enables use to seek out organisms that
already utilize efficient enzymes for the breakdown of
cellulose into fermentable glucose.

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Bioprospectors are really looking for the enzymes produced by
organisms for more efficient pretreatment & hydrolysis
Once an organism is found, it must be selectively plated,
isolated, screened, and tested on different feedstocks.
Genetically Engineered Bacteria Could
Lead to Cheaper Cellulosic Ethanol

http://current.com
/news/89289090_g
eneticallyengineeredbacteria-couldlead-to-cheapercellulosicethanol.htm