Enzymes, Ethanol, and Biofuels...Oh My!

Introduction

A: Background Information
          The term "biofuels" refers to a group of fuels that are produced from biological sources that were recently living. This is in contrast of fossil fuels which have been created by biological sources long dead. Biofuel technology now-a-days consist of four catigories: cellulosic ethanol, ethanol production from sugar and starch sources, syngases, and biodiesels. In this lab we will be working with cellulosic ethanol which is the break down of cellulose to glucose followed by a fermentation step to ethanol. However, we will only go from cellulose to cellobiase. Enzymes are very important in this lab as well. They speed up the reaction by positioning the substrate in a specific way so that the transition state of the reaction is saturated. The enzyme in this lab is cellobiase. It breaks down the two glucose molecules connected by a glucoside linkage. In addition, the substrate of the lab is cellobiose. In summery, this lab works by having cellulose and using enzymes such as cellubiase to break apart the two glucose molecules which are connected. This creates two separate glucose molecules which can create fuel. This fuel is then used for energy (ATP) and can be converted to ethanol. One can see the products of our lab by using the stop solution, which is a strong base, to kill the cellobiase as the P. Nitrate made from the glucose turns more yellow as more product is created.
 
B: Objective & Purpose
          The objective of this lab is to study the reaction rate of cellobiase (the enzyme used to break down and turn cellobiose to glucose). By studying all those, we will also analyze how temperature, pH, substrate concentration, and enzyme concentration affects the activity of the cellobiase. The lab is also used to test the ability of a comment fungus (mushroom), to break down the provided substrates and determine how efficient they are. The purpose is that many biofuel industries around the world are currently trying to find a natural source of cellulose enzymes that will be more stable at extreme conditions for degrading plant source cellulose. This is a more natural and helpful way (ethanol) to create energy and not use petroleum or gas as a major energy source. 
 
C: Procedures
Day 1:
          First thing one must do on day 1 is find the four 15 ml conical tubes labeled: "Stop Solution", "1.5 mM Substrate", "Enzyme", and "Buffer". Afterward, get the seven cuvettes from the front desk and label five cuvettes E1-E5, and the remaining two cuvettes "Start" and "End". Then using a sterile DPTP, pipet 500 ul of the Stop Solution into all of the cuvettes and clean the DPTP well with water. Label the two empty 15 ml conical tube "Enzyme Reaction" and "Control". When done, use the clean DPTP and pipet 2 ml of the 1.5 mM Substrate into the 15 ml conical tube labeled "Enzyme Reaction". Then use the same DTPT to pipet 1 ml of the 1.5 mM Substrate into the "Control" conical tube and rise the DPTP well with water. Afterward, label one DPTP "E" for enzyme to only use it with the Enzyme Reaction Tube, and another DPTP "C" for control to use only for the Control Reactoin Tube. Then using the "C" DPTP, pipet 500 ul into the 15 ml into the "Control" conical tube and gently mix. Once mixed, remove 500 ul of the solution with the same DPTP and add it to the cuvette labeled "Start". Then use the "E" DPTP, and pipet 1 ml into the "Enzyme Reaction" conical tube and gently mix. START YOUR TIMER! Now use the "E" DPTP again and remove 500 ul of the "Enzyme Reaction" and add it to the cuvettes labeled "E1-E5". E1 should have the solution added after one minute, E2 after two minutes, E3 after four minutes, E4 after six minutes, and E5 after eight minutes. Once all those cuvettes are filled, use the DPTP labeled "C" to remove 500 ul of the "Control" reaction tube and add it to the "End" labeled cuvette. Finish this lab by analyzing your samples and rising out the reaction tubs and the cuvettes, throwing away the DPTPs and cleaning your lab station!

Day 2:
          Start the lab by weighing out one gram of mushroom and putting it into a mortar with 2 ml of extraction buffer, and using a pestle, grind the mushroom to produce a slurry. Then using the centrifuge at the lab table, scoop 1.5 ml of the slurry into a microcentrifuge and place it inside the centrifuge for two minutes to pellet the solid particles. Once that is done, get cuvettes from the teacher and label them "1-5" at the top of the cuvette. Then use a clean DPTP to pipet 500 ul of the stop solution into each of the five cuvettes, then rise out the DPTP with water. Afterward, label the empty 15 ml conical tube with the type of mushroom you are using and use the clean DPTP to pipet 3 ml of substrate into the tube. Then use a new DPTP and pipet 250 ul of the enzyme extract (mushroom extract) into the conical tube labeled with the mushrooms name and contains the substrate. START YOUR TIMER! Remove 500 ul of the mushroom extract and substrate mixture from the reaction tube and add it to all five of the cuvettes. For cuvette 1 wait one minute, cuvette 2 wait two mintutes, cuvette 3 wait four mintues, cuvette 4 wait six minutes, and cuvette 5 wait eight minutes. Once done, proceed with the analysis of the lab and rinse out all cuvettes and reaction tubes, throw all used DPTPs away and make sure lab stations are clean!

D: Controls, Variables & Prediction

          The control of this lab is the "Control Reaction" tube which contains only cellobiose and buffer. The variables of the lab are all the different cuvettes used throughout each lab to test the amount of product. My prediction of this lab will be that as all of the solutions are added to the cuvettes, it will create P. Nitrate that will yellow when the reactions of the solutions become stronger. So by waiting longer and longer for each of the cuvettes when we add solutions to them, they will become more yellow, which means the product (glucose) will be more and there will be more fuel/energy.


Observations: 

Day 1:

  

Day 2:

Discussion:

A: Analyze and Explain Data
Day 1: 
          My lab table and I began our lab on day 1 by reading all the directions and going through the procedures as they were written; everything went extremely well. Our results/data that we acquired from the lab were what we hypothesized before we began the lab: the color of the P. Nitrate will become more yellow as time goes on, proving that the solution created more energy as time progressed. The color in the cuvettes turned more and more yellow as added the enzyme reaction as time progressed. This proved that our data was correct and we did the lab correctly, we were able to create a biofuel!
Day 2:
          My lab table and I began our lab on day 2 by reading all the directions and going through the procedures as they were written; everything went extremely well. Our results/data that we acquired from the lab were what we hypothesized from the day before and before we began the lab: the color of the P. Nitrate will become more yellow as time goes on, proving that the solution created more energy as time progressed (even with a mushroom used). Although the color of the P. Nitrate was not as bright as the previous day, the color still become more and more yellow as time progressed. This showed us that even with a fungus, one may still acquire the same results in a different way. This proved that our data was correct and we did the lab correctly, we were able to create a biofuel from a fungus!

B: Possible Sources of Error
          Some possible sources of error in this lab can be if the "Enzyme reaction" or "Control" was not mixed in the lab on day 1. This would cause a not as preside result when adding them to the cuvettes. A source of error which could happen on day 2 is if the slurry mushroom solutions was not left in the centrifuge for the two minutes which it was suppose to be left in. In addition, a source of error which could happen throughout both days of the labs is if the materials, such as the DPTPs, the conical tubs or the cuvettes had not been cleaned out properly. Also if one did wait the exact amount of time they were asked to wait before adding a solution to each of the cuvettes. That would be an error because if all supplies are not cleaned properly, it will contaminate different parts of the lab and the lab results will not be correct. This could be huge error because the purpose of the lab is to wait and compare all the cuvettes and since one did wait the amount of time they were asked, their results will be incorrect.