1. Using Cyanobacteria to Increase Yield of Ethanol in Plants By: Vishal Mummareddy

2. Ethanol Production in the U.S. Americans used 13 billion gallons of Ethanol last year World Ethanol production has tripled from 2000 to 2007 36 billion gallons of Ethanol will be produced during the year 2022

3. How Ethanol is Made?

4. Cellulose and Hemicellulose Cellulose and Hemicellulose are the sugars used to make ethanol They are found in the cell wall of most plant cells Lignin is intertwined with cellulose and hemicellulose and must be seperated in order to make ethanol Cellulose and Hemicellulose are made of thousands of glucose units So if we can increase glucose production, we can increase cellulose production which will then create more ethanol

5. Cellulose and Hemicellulose

6. Plant Cells Many plant pathogens infect plants through the roots, but some of them can get in through the stomata The stomata is used for gas exhange and opens when there are changes in the environment The purpose of these bacteria are to colonize the plant and eventualy kill the plant Pseudomonas syringae is a bacterial plant pathogen that can go through the stomata of plant leaves

7. Pseudomonas syringae Is an airborne bacteria that infects many different types of plants It causes the water in plants to freeze which results in injury for the plants

8. Cyanobacteria Cyanobacteria is one of the most productive photosynthetic bacteriums This type of bacteria can be found in almost every terrestial and aquatic habitat For my design project, I am going to use cyanobacteria and the components of Pseudomonas syringae to infultrate plant leaves and increase glucose production

9. Getting Cyanobacteria into Plants The leaf of plants is a great place for bacteria to grow and replicate because: 1. Moist 2. Sunlight In order for cyanobacteria to invade the plant it must release virulence effector proteins into the plant

10. Getting Cyanobacteria into Plants To trigger the proteins, the farmer must spray Gibberellic Acid: a growth hormone for plants This hormone will then release the virulence effector proteins in the cyanobacteria which will then trick the plant into letting in the cyanobacteria These proteins can be found in Pseudomonas syringae Then the bacteria will grow into the plant and travel to the spongy mesophyl

11. Plant Leaf

12. Cyanobacteria Entering Plant: Stage 1

13. Cyanobacteria Entering Plant: Stage 2

15. Cyanobacteria Entering Plant: Stage 3

16. Cyanobacteria in Plant Leaf The cyanobacteria will connect to the plant cell using the hrp- gene cluster. This gene contains a needle that extends from the cyanobacteria This needle is used to transport proteins that will infect the host cell However, once all the proteins are taken out, it can be used to export glucose Size of protein - 3 – 6 nm Size of glucose molecule – 1 nm

17. Cyanobacteria Joining with Plant Cell

19. Engineering Cyanobacteria The first step of the proccess would be to get the bacteria into the plant To get this done, cyanobacteria needs to secrete virulence effector proteins The effector protein tricks the stomata into letting the bacteria inside

20. Engineering Cyanobacteria In addition to the virulence effector proteins, the cyanobacteria needs a hrp-gene cluster This gene cluster contains the coding for a base, needle, and tip wich can be used to transfer molecules into the host cell

21. Engineering Cyanobacteria

22. Expected Results The bacteria should release glucose using the chaperone molecules when it is conjoined with the plant cell Conjoined with Plant CellTransfer Glucose 00 11

23. Making Ethanol Now that we have a bacteria that can make and transport glucose into the plant, we can then increase the yield of ethanol This happens because the plant cells store the extra glucose in forms of starch in the leaves, roots, or stems

24. Making Ethanol After the crops are harvested, it undergoes 5 major processes in order to make ethanol 1.Milling 2.Liquefaction 3.Saccharification 4.Fermentation 5.Distillation and Recovery

25. Milling This is when the crop is smashed in order to get it into its basic components

26. Liquefaction Is when the smashed crop is heated and the enzymes break down the starches into smaller fragments

27. Saccharification The starches are then broken down into glucose

28. Fermentation Yeast is then added to the glucose to start the process of converting glucose into ethanol

29. Distillation and Recovery The ethanol is then removed and used for fuel

30. Advantages My design will always be able to increase glucose production because most companies focus on making the plant more productive. But my design will always work and act as and additive for glucose production

31. Advantages This design will help increase the yield of ethanol production in plants. This will definitely make ethanol more economical. As a result, more consumers will start to use ethanol.

32. Potential Problems Some bacteria may not be able to enter the plant or may die in the mesophyll The bacteria could mutate which can lead to the bacterium harming the plant The bacteria could travel and infect other plants and harm other crops

33. Testing I would test the bacteria on plants in a lab setting and measure the production of ethanol from a plant with my bacteria and one without it. This will prove that my bacterium is effective and can increase the yield of ethanol in plants.

34. Works Cited http://www.ncbi.nlm.nih.gov/pubmed/18077204 http://www.unl.edu/alfanolab/pdf/publications/14.pdf http://en.wikipedia.org/wiki/Pseudomonas_syringae http://www.pnas.org/content/97/16/8770.abstract http://www.sciencedirect.com/science/article/pii/S0092867406010154 http://www.ncbi.nlm.nih.gov/pubmed/17419713 http://www.ncbi.nlm.nih.gov/pubmed/16439654 http://www.extension.purdue.edu/extmedia/ID/ID-328.pdf

35. Works Cited http://faculty.mville.edu/ParikhS/courses/chm3049/articles/Defossiling%20F uel%20- %20How%20Synthetic%20Biology%20Can%20Transform%20Biofuel%20Produc tion.pdf http://faculty.mville.edu/ParikhS/courses/chm3049/articles/Defossiling%20F uel%20- %20How%20Synthetic%20Biology%20Can%20Transform%20Biofuel%20Produc tion.pdf http://www.epa.gov/ttnecas1/regdata/IPs/Starch%20Manufacturing_IP.pdf http://www.ncbi.nlm.nih.gov/pubmed/18476862 http://biofuelsandthepoor.com/facts-and-definitions/ http://www.sciencedirect.com/science/article/pii/S0958166908001420 http://www.icgeb.org/yazdani-shams-lab.html http://www.triplepundit.com/2012/01/doe-lab-synthetic-biology-produce- cheaper-biofuel/ http://www.triplepundit.com/2012/01/doe-lab-synthetic-biology-produce- cheaper-biofuel/