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Summer 2012 Blog - Akshita Dutta

Akshita Dutta is spending eight weeks at the Indian Institute of Technology, Kharagpur.

August 20, 2012

As I conclude my work here, I want to reflect on my time here and the impact this internship has had on me academically and culturally. I had minimal knowledge of microbial biology and enzyme production before I arrived at IIT. In the period of two months, however, I’m can  understand some of the most esoteric literature on this topic. And I have my faculty advisors, my lab colleagues, and especially my mentor to thank for this. They have been very supporting and helpful throughout my time here. It gave me great pleasure to have been influenced by them.

 akshita_lab_teamLab Team

As I depart for home next Friday, it will be very difficult to say goodbye.  I have become more independent and grown as a scientist during the Cal Energy Corps internship. It’s something I know I will take back with me to Berkeley. In the past seven weeks, also, I’ve travelled around India and learned to appreciate its colorful culture. Lastly, I want to thank Graham Fleming and Cal Energy Corps for this invaluable and memorable opportunity.

August 3, 2012

With my time at IIT coming to a closure, I will be spending my last week wrapping up my project and presenting it at a symposium arranged by our faculty advisors. This also means that I can provide with you a more thorough overview of the work I have been doing since my last post! Since my last update, we have successfully optimized the delignification process based on parameters such as temperature, incubation time, and substrate concentration. The motivation for optimizing the process is that if we chose to upscale this process for industrial purposes, the most efficient operating parameters can be employed to give the highest yield.  The competence of the process was identified by measuring the amount of lignin remaining after delignification using a titrative method. We further characterized our substrate post- processing using XRD, FTIR, and SEM to observe changes in crystallinity and retention of its basic chemical structure.

We then moved on to the enzymatic hydrolysis step of the bioethanol production procedure. As before, our initial goal was to optimize for the parameters and reasons mentioned above. We then moved on to understanding the intrinsic kinetics of this step and this proved to be more of a challenge than initially expected. The enzyme we used for this process is known as cellulase which was extracted from a species known as Trichoderma Reesi. It is commonly used in microbial biology and so was available to us for inoculation and growth. Crude cellulase itself is composed of various different enzymes that work together to breakdown cellulose and hemicellulose into smaller sugar molecules that can be processed into ethanol by fermentation. Previous kinetic modeling studies on lignocellulosic substrates have focused on using pure enzymes during experimentation since it is easier to untangle the effects of each enzyme on the substrate this way. So the simultaneous presence of these enzymes makes it difficult to understand the nature of the reaction and hence, accurately measure the governing parameters. Faced with this obstacle, we are attempting to identify the types of inhibition active in our system and we have made some progress in this but more work can be done to corroborate our results. I will conclude this post here with a promise of another one next week that will include pictures from our symposium and some of my reflections on the past few weeks!

July 16, 2012

I arrived in Kharagpur exactly a month ago and this past month has been an invaluable experience, both academically and culturally (but more on that some other time). My project, which is supervised by Professor Rintu Banerjee and Professor Saikat Chakraborty, is carried out at the Department of Food and Agriculture. The main focus of my work here is to produce bioethanol from a viable substrate which in this case is Cotton Stalk. Let me backtrack a little to explain why this substrate was chosen in the first place.

India’s growing population and scarcity of arable land have been important concerns for many years. As you are probably aware, bioethanol production depends on using crops to convert sugars into a fuel source. At a glance one can see the possible competition that might arise between food and fuel sources if one wanted to use this process for fuel production on a large scale. It is this concern which motivated us to choose Cotton Stalk as the substrate for bioethanol production. It is an abundant and non- edible crop and hence, perfect for the project!

Having completed my 3rd year in Chemical Engineering with a concentration in Materials Science, my only previous background in plant biology consisted of the AP Biology course I took in high school. Needless to say, that first day that I met Professor Rintu Banerjee and the rest of her laboratory was daunting, especially when words like lignin content and hemicellulose were casually thrown into conversation.  In a matter of minutes though, I learned that my anxiety was superfluous because my mentor spent some time helping me to familiarize myself with the basic concepts and processes associated with bioethanol production. And here is my chance to relay the information to you as it was taught to me. Those who are better versed in biology and ethanol production should excuse the next few paragraphs!

The secondary cell wall of plants is composed of, among other things, lignin, cellulose, and hemicelluloses. Lignin is a polymer of phenol groups and an integral part of cell walls. It cross- links with hemicellulose which is composed of various sugars. Cellulose is a crystalline structure composed of polymers of glucose. Since we want to convert sugars to ethanol and lignin is notably lacking in it, we want to get rid of it! And this leads us into the specifics of ethanol production.

Lignin is eliminated through deligninfication (also known as pretreatment) which is followed by saccharification and fermentation (or sometimes, simultaneous saccharification and fermentation, SSF). Saccharification is the process of degrading cellulose and hemicellulose to access glucose and the monomeric sugars. Fermentation then translates these sugars into ethanol. SSF combines these two steps in an attempt to make the process more efficient. This is possible because the product of saccharification does not inhibit the fermentation process.

Before any of these steps can be carried out however, we need to characterize our substrate in terms of lignin, protein, reduced sugar content, etc. It is favorable for the substrate in use to have a smaller amount of lignin since it is discarded. After characterization we concluded that cotton stalk was indeed a promising source for bioethanol production. Since then, we have worked on optimizing the operating conditions of deliginification and saccharification. The next half of my internship will focus on modeling the kinetics of the saccharification stage of this process and comparing it to the model produced for SSF. I will elaborate more on the techniques we have using for characterization and the lab atmosphere more in my next blog post so stay tuned!

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