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) A study conducted by the USDA and DOE found that one billion tons of biomass exists in the U.S., and that if this billion tons of biomass is converted into biofuels, the U.S. can reduce fossil fuel use by 30%. 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.

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. Pretreatment is an industrial process using acid, ammonia, high heat and pressure. The organisms in the student samples must not only carry out the hydrolysis of cellulose, they must first perform a natural pretreatment to remove lignin and hemicellulose. This is why there is often a community of multiple microbe species working together to degrade material. Goal of pretreatment is to open up cell wall and expose cellulose. www.glbrc.org

Hydrolysis with cellulase Cellulose must be broken into individual glucose molecules using cellulase enzymes before fermentation can occur. glucose cellulose enzyme glucose www.glbrc.org

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.

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. 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 From Currie, C. 2001. Annual Reviews of Microbiology, Created by Cara Gibson and adapted by Angie Fox

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_g enetically- engineered- bacteria-could- lead-to-cheaper- cellulosic- ethanol.htm