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.

*Based on U.S. Energy Consumption 2004

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

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.

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Year 2008 2010 2012 2014 2016 2018 2020

Other

Biodiesel

Cellulosic

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Biofuel Year: 2007 2010 2022Other 0.20 4

Biodiesel 0.65 1

Cellulosic 0.1 16

Corn Ethanol 4.7 12 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. 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 cellulaseCellulose must be broken into individual glucose

molecules using cellulase enzymes before fermentation can occur.

glucose

cellulose

enzyme

glucose glucose glucose

Improving Biomass Pretreatment A key goal for improving pretreatment strategies is to

discover and improve natural cellulose-degrading enzymes 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.

Genome Management Information System,

Oak Ridge National Laboratory Http://genomics.energy.gov

BIO-PROSPECTING

Bioprospecting can range from trekking across the Amazon to trekking across the street to a cow pasture.

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. 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.

Leaf-cutter Ant Symbiosis

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Video 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. An enzyme is really just a protein that speeds up

or slows down a chemical reaction. Enzymes have two key components:

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”). 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 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. 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. 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 Once a microbe has been recovered…

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 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. 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_genetically-engineered-bacteria-could-lead-to-cheaper-cellulosic-ethanol.htm