Jennifer Jong is spending ten weeks in Brazil at the Brazilian Bioethanol Lab.
June 18, 2012
Separating sugar cane tissues
I am in Campinas, Brazil at the Brazilian Bioethanol Science and Technology Laboratory to study the structure of sugarcane cell walls. My supervisor developed this technique of using thermoporometry and sorption kinetics to study cell wall structure. The cell wall is made of different components all intertwined and meshed together. They form little pores within the cell wall.
Thermoporometry measures the rate at which water within these pores melt. Sorption kinetics measures how fast water is sorbed into the crystals surrounding the walls. Through a series of calculations, the pore size and cell wall structure can be determined. My supervisor developed this technique using processed cellulose and other materials with defined pore sizes. My project will be to pick two different areas of the sugarcane and to examine the cell walls. Knowing and understanding the cell wall structure will help know what type of enzymes will be able to degrade different areas of the cell wall.
Brazil is a country that relies heavily on ethanol production. It is interesting to see most cars in Brazil are flex fuel cars. This means that they can take gasoline or ethanol. Gas stations only have two prices; the price of ethanol and the price of gasoline. To be competitive, ethanol has to be 70% of the price of gasoline because ethanol doesn't produce as much power, but recently, due to some political decisions, gasoline has been cheaper. This made me realize that a lot of being environmentally green has to do with government policies. I had not realized the importance of this before.
I must admit that I was extremely apprehensive about the weather here in Brazil before I left the United States. It is wintertime in Brazil, but the weather is surprisingly nice. Warm sun and dry air. Instead of being concerned with the weather, I should have been worrying about learning Portuguese. Outside of my lab, everybody speaks little English, but in my free time I try to cram Portuguese vocabulary.
July 7, 2012
Sugarcane stalk cross section
As I previously mentioned, the main source of fuel in Brazil is ethanol from sugarcane. Sugarcane is harvested from the fields, and then sent to the mill to squeeze out the sugars that are then fermented into ethanol. What remain of the sugarcane stalk are mainly the cell walls, known as bagasse. Most bagasse is burned in mills to provide electricity. Bagasse is rich in cellulose, byproducts of which can be fermented. However, cellulose is difficult to access because it’s intertwined with lignin and hemicellulose to give structure to the cell wall. Research is being done to find an efficient and economical method to extract this cellulose.
The objective of this research on pore sizes is discovering more about what makes up the majority of bagasse, an area relatively unknown. Understanding where each part of the bagasse comes from the sugarcane will help focus the research on bagasse treatment. To determine the components of the bagasse, fresh sugarcane can be separated into its different tissues and sent to thermoporometric analysis. Through thermoporometric analysis, the characteristics of the different tissue cell walls can be determined and compared with the cell walls of the bagasse. For my project, I have decided to look at the different tissues of the sugarcane, specifically the parenchyma, the vascular bundles and rind. The parenchyma is the soft spongy tissue that fills most of the plant. Its main purpose is to store the sucrose in sugarcane. Vascular bundles are used to transport water and nutrients through out the cell and makes the sugarcane very fibrous. The rind is the outer part of the sugarcane and functions to keep the sugarcane upright. It consists of immature bundles and parenchyma.
This research on pore size can also be applied in the future. Sugarcane is a very versatile. Although its primary function now is for ethanol, people are looking to making it a multi-industry crop and to specialize different parts of the sugarcane for different industries. Different characteristics, such as pore size, can give rise to different properties, which can lead to specialization.
A couple of test tubes with samples
After a month of gathering sugarcane samples and separating parenchyma from vascular bundles from rind, and starting thermoporometry, results show that parenchyma -- as expected -- is able to hold more water in its pores than rind and vascular bundle. Although thermoporometry measures the amount of water melting in pores, however, it doesn't distinguish where water is melting. The tissue is fresh, not squeezed and processed as is bagasse; therefore, pores can be formed by many structures within the cell. We limit machine measurements within the expected pore sizes (for example, it won't measure plasmodesmata pores many times bigger than the expected pore size; but the machine will still measure pores of structures similar to pores of the cell wall). One structure that possibly interferes with results is the cytoskeleton. Though it has a small mass compared to the cell wall, it may hold a considerable amount of water. I'm currently working to mediate this problem through a pre-treatment of proteases.
July 31, 2012
Air drying samples
Cytoskeleton removal is harder than anticipated. An important part of solid state cell wall analysis is to keep the cell wall intact as much as possible to examine the pores. Most cytoskeleton removal protocols require grinding of the plant material to break the cells as much as possible. This is to increase access to the cell material within the cell wall. In an attempt to keep the cell wall intact as possible, I soaked the plant material protease and buffer in hopes that the protease will work its way through the cell wall. I set up different conditions to see if the protease digested any proteins. Bradford experiments were set up to show the amount of proteins in the solution. Results show that no proteins were digested.
The big lesson that I learned from this cytoskeleton removal side project is that research is hard. It’s not just pipetting and doing PCRs. As an undergrad in my previous labs, I’d just receive instruction from my post-doc or grad student. Having control over your own project is very different. Looking for papers and protocols that would hopefully just fit your project is difficult. However, I learned that it is important to keep focused and motivated. Overall, it was interesting to get to ask the questions.
Another part of my project is to look at alterations to the cell wall. There are different methods to preparing the cell wall for analysis. Some protocols require the plant material to be air dried, oven dried at 100°C, heated in 100°C water or soaked in 80% ethanol. These conditions may alter the structure of the cell wall which means analyses that use these preparations may not be measuring the actual state of the cell wall.
August 10, 2012
Vascular bundle cross section of a sugarcane
The sorption part of my project is now completed. Sorption measures the water in the pores that are in contact with the surface. The amount of water in contact with the surface changes based on hydrophobicity and mass of the sample. Thus, sorption can help us understand cell wall composition in different tissues located at different areas of the cane. Results seem to show little variation between tissues and location. There is a small variation in composition in tissues from the top internode as compared to the bottom internode. This is understandable because the top internode is still developing and should have high variability.
The alteration of cell wall experiments is almost completed. I ran into difficulty preparing samples for these experiments. I had less tissue than expected and had to separate more. This led to tissue coming from different areas of the internode meaning precision of my data decreased. I also forgot to prepare some samples for a control experiment. Since I only have two more days here and I ran out of tissue samples, I’ll just have to make do with my data.
As a supplement to my project, I was to perform monosaccharide analysis on my tissue samples at the University of São Paulo. A monosaccharide analysis is a more detailed analysis of the composition of the cell walls and was to supplement my sorption data. But I’ve run out of time. The samples are already prepared and will be analyzed after I have left.
At the Catedral da Sé, São Paulo with fellow Cal Energy Corps
participants: Susan, Joanna and Hank.
I can’t end this last blog post without saying thank you. Thank you to Cal Energy for giving me this wonderful opportunity. I have learned a lot and gained many valuable experiences. Thanks to Carlos, my supervisor, for teaching me all the intricacies of research. I also have to thank the other interns in Brasil. Thanks to Joanna, Hank and Susan for making my already amazing summer in Brasil many times more awesome.