Creating Biofuels from Waste

Creating Biofuels from Waste
Purpose
Biofuels mark a great step into today?s efforts to slow down global warming. However, when food sources, such as corn and soy are used as derivatives for ethanol, the impact is far worse than using fossil fuels. Biofuels require more energy to do artificial refinery, cultivating, and collecting; whereas, fossil fuels already meet all the prerequisites through millions of years in the earth, and thus use less energy. On the social justice stance, the poor suffer through the sky-rocketed food prices because farmers are using their food to supply the ethanol demand. As technology becomes more efficient, society can rely on a source of energy that deters global warming, uses less energy to process, and betters the world economy.
Question
Which specific cellulase from fungi breaks down bleached paper waste efficiently?
Why?
Bleached paper is solely composed of cellulose (20-25%) which it makes it an ideal source for fuel because its lack in lignin makes it more efficient in the refinery process.
Fungi serve as vital decomposers in the natural world. By channeling this idea to break down paper waste into sugars, the possibilities are endless.
Terms to know
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Cellulase are enzymes that break down cellulose. Fungi and bacteria have special enzymes that are designed to decompose material.
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His-tag is a tag that helps the identification process by attaching to a protein. A His-tag is a small tag that aids the purification process by binding with a nickel nta matrix.
Materials
-Agar plates
-Sterile q-tips
-Yeast
-Incubator
-Bleached paper waste
-Fungi
-pcr
-Primers
-Blue dye
-Plasmid: pTrcHis-Topo
-Wash buffer
-Elution buffer
-LB broth
-E.Coli competent cells
-Ice
-Water
-Filter
Method:To insert the desired sequence into the plasmid
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Use a fungi that carries the right dna sequence to break down cellulose, for example Trichoderma viride(T. viride)
-Fungi:are eukaryotic and heterotrophic. Fungi have cell walls made of chitin and can be decomposers, antibiotics, pathogens, and foods.
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Make a primer to attach to the specific wanted sequence
-pcr primers: block out the sides of the specific dna sequence that wants to be copied.
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pcr the dna sequence to make copies
-pcr:also known as, Polymerase chain reaction. pcr is a process that allows a specific sequence in a dna to be copied. Scientists use this to further their research.
Method:To insert the dna into the plasmid
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Add the purified pcr product into the microfuge tube
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Add the pTrcHis-topo
-Plasmid: are round dna found in eukaryotes and prokaryotes. Scientists utilize plasmids to put in other kinds dna into cells.
-pTrcHis-Topo:is a plasmid that makes proteins that can be purified with nickel.
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Incubate
Method:Inserting the plasmid into E.coli
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Put the plasmid reaction in with the E.colicompetent cells together in a microfuge tube
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Incubate on ice and heat shock it in 42 ?C water and put it back into the ice bucket
-Chemically competent cells:After shocking cells, they absorb the surrounding dna.
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Spread the solution onto the agar plate containing nutrients and ampicillin
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Incubate at 37?C to cultivate E.coli.
Method:To grow into a liquid culture
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Using a pipette tip, pick a white colony and drop it into a microfuge tube with LB broth and incubate at 37?C while shaking
Method:Purifying Process
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Spin the solution
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In a microfuge tube, re-suspend the pellet in wash buffer
-Wash buffer:allows the His-tag protein to bind and all other by-products to be washed out, so all that is left are the protein and column. This solution contains 50 mM Sodium Phosphate (as a buffer), 200 mM NaCl (prevents proteins from sticking together and keeps them stable), 20 mM imidazole (prevents unwanted binding)
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Sonicate
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Spin
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Collect the supernatant into a nickel column
-Supernatant:liquid surrounding a pellet in a microfuge tube
Method:Purifying Process
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Put wash buffer and elution buffer through
-Elution buffer:has more imidazole (500mM) that washes the protein off the column for collection
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Result = Purified protein!!!!!
For final testing:Cellulase Activity Measurement
A cellulase assay tests if the enzyme truly breaks down the cellulose.
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Cellulose and a blue dye bond are suspended in water. This solution is insoluble because it is a large chain of glucose and blue dye.
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When the cellulase enzyme is added it will break down the chain into individual, soluble dye and glucose.
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This is filtered and if the supernatant shows blue dye, the enzyme is breaking down cellulose.
Current Problem:
This is a slower process and is difficult to apply at the industrial level especially when the demand for Biofuels and energy is so high.
With Additional Research:
With additional research, scientists can ferment the sugars into alcohol and thus, make Biofuels at the industrial level. Additional research can explore the ability of other microorganisms to break down cellulose more efficiently.

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