After countless weeks of dehulling kudzu pods to get enough seeds, the time has finally come to extract the oil (Our goal is to extract oil from kudzu seeds and produce biodiesel from it, we will then determine the feasibility of using this biodiesel). We attempted to use a Burr Coffee hand grinder to grind the seeds but spent the majority of time having difficulties with the grinder and ended up using an electrical grinder instead. After the grinding process was completed, we ultrasonicated the ground up seeds with hexane (a chemical: it’s pretty dangerous) to release the oils. The rotary evaporator was used (a fancy device that evaporates the hexane from the oil, so you’re left with just oil). We came back to the wonderful sight of kudzu oil! .09 grams of translucent green beauty. We plan to extract the oil that is left in our ground seeds in the future.
After spending weeks de-podding our abundance of harvest kudzu seeds, we recently finished running 2000 kudzu seeds mixed with hexane through the rotary evaporator. The rotary evaporator separates the hexane from the kudzu oil. We also ran a test trial using some of our ground up soybeans. The 2000 kudzu seeds produced 0.09 grams of oil.
We have oil! Thats right REAL kudzu seed oil. It took a long time and a lot of dehulling. But we did it!
We recently put the oil and hexane mixture into the rotary evaporator to remove the hexane. The rotary evaporator works by heating the mixture and creating a vacuum, causing the mixture ti evaporate. Once the mixture evaporates the hexane condenses on the coils and then is collected in a flask for reuse. We have been playing with the conditions some to find the optimal conditions for the hexane to evaporate. The lower the pressure the higher the temperature the faster the hexane will evaporate. However disaster can occur if temperature is too high or pressure is too low. Wish us luck!
As our (Bain Brown, John Nguyen and Nicole Rideout’s) project is getting started we have decided to do a little bit of practice. The best part you ask? The rotary evaporator!
So, what is a rotary evaprator, well the Rot. Evap. is the the machine that seperates a mixture into its parts (in our case, hexane and the oil). It works by creating a vacuum, bringing the mixture to a temperature where the desired substance will evaporate, and then condensing and collecting the evaporated liquid in a different flask. This leaving the mixture separated in 2 different flasks. See diagram below
Why is it exciting? It shows progress! Finally being able to see our oil left in a round bottom flask is a major confidence booster. Our project finally feels like it is going somewhere! We will eventually be using the Rot. Evap. to separate the kudzu oil form the solvent (hexane), but for now we are only using it to practice. Check out the video to see it in “action. Video of Rotary Evaporator
Our group, BAJONI (BAin, JOhn, NIcole), is working on a practice solvent extraction trial on soybeans in preparation for the kudzu solvent oil extraction. Kudzu as you may have encountered on multiple occasions, is an invasive plant native to Asia. It was introduced to the US in 1876 at the Phillidalphia Centennial Exposition. Farmers were then encouraged to plant this vine from the 1935s to mid-1950s to stop soil erosion. Kudzu is now found in 30 states in the US and is causing environmental conflicts. Instead of trying to get rid of this vine, we are finding a way to take advantage of its vigorous growth rate. We are currently working out methods to extract oil from kudzu seeds. The oil will be turned into biodiesel. We will then determine the feasibility of using kudzu seed oil as a source of biodiesel.
My group (including Bain Brown and John Nguyen)’s project is determining the feasibility of using seed oil from kudzu as a potential for biodiesel. Kudzu seeds are normally mature and ready to harvest at around mid-October, meaning we’ll need to start harvesting soon. There is a possibility that the kudzu vines at the school near the football field won’t have enough seeds to extract enough oil, and that we may need another source, but we’ll soon find out!
The search for lignin degrading enzymes is starting to come to a close. After identifying which of our samples were producing enzymes using the guaiacol assay, we performed PCR on these samples and sent the isolated DNA of to be sequenced. After receiving the sequenced DNA, we blasted the samples using the GENBANK database. Our results were surprising. Apparently, the green mold we had thought was contamination was actually a LDE producing fungi! I guess we should have done some more research on the fungi we had assumed was hurting our samples before we jumped to conclusions.
Now that we have identified our samples, were are moving into the project assembly phase. Much of the past few weeks had been spent creating our final presentation, writing our paper, and creating graphs. With Christmas break coming and our project coming to close, our group is experiencing the bittersweet feeling of the end coming into sight.
Recently, Jolizen has finally isolated an enzyme! We isolated cellobiase, an enzyme that breaks down a type of cellulose, We visited a local lab, called PharmAgra, that does pharmaceutical research. They have larger centrifuges available for our use. We took our fungus over after we added ammonium sulfate that was used to make the proteins a solid. After it was spun at 900 rotations per minute, the proteins all stuck to one side of the bottle. We tested the proteins, and they tested positive to be cellobiase! We could tell because when cellobiase is present, the solution turns yellow.
Over the past week, the Jolizen station has been quite colorful. Screening methods using dyes have been used to detect enzyme production. Originally, we were using dyed plates and looking for a change in color as the fungus grew on the plate over the period of one week or more. After unclear results with this type of method, a new idea for enzyme detection was adopted. After growing our fungus for a few days, the plates are then flooded with the dye and results are available in 10 minutes, rather than 1 week, and are much more definite. The first assay of this kind detected amylase, an enzyme produced in our saliva that breaks down starch. Our fungus was grown on potato dextrose and flooded with Gram’s iodine. The starch that had not been “digested” stained black, leaving a ring of clear fungal growth where our fungus had broken down the starch. Amylase is being produced! The second assay was stolen from Hannah and Ryan and was the answer to our prayers! Our fungus was grown on LDE plates. When we returned from the weekend, the fungus an the surrounding quarter inch were brown to orange in color while the rest of our plate was clear. This indicates the production of ligninolytic enzymes, or enzymes that break down lignin. Our last assay which detects for production of cellulases (they break down cellulose) will happen tomorrow. Our fungus has been growing on cellulose and will be flooded with Congo red dye. Let’s cross our fingers that it’s 3 for 3!
The pressure cooker has been running nonstop this past week in the TIME room, cooking every type of agar known to man. Our refrigerator is stocked full of potato dextrose, blue, red, and water agar, so come and get ’em! Yesterday and today, the infamous “Jolizen” team (made of members Eliza, Joe, and Lauren) has been transferring our fungus, Diaporthe sp., to plates filled with 3 different types of mediums. The red plates (making up the red stripes of our homeland’s flag) have cellulose and a special Congo Red dye (especially extracted and shipped to us on the back of African elephants straight from the heart of the Congo). If this dye shows discoloration by the start of next week, our fungus is producing some type of cellulase enzyme. We also are growing our fungus on blue plates (found in the blue star corner of the flag) which, if color change occurs, will indicate activity of an enzyme that breaks down lignin. Lastly, we cultured on potato dextrose agar (the white stars and stripes) for the purpose of maintaining a healthy base line fungus to take isolates from. We performed DNA extraction and PCR on these PDA cultures last week so that we can be sure they are still Diaporthe sp. Stay tuned for the results of our colorful tests!