The blame goes to my project for the strong smell of garlic floating around in the TIME room this semester. The lab smells like garlic because I’m testing the antifungal effectiveness of garlic and four other plant extracts (goldenseal, echinacea, and olive) on two fungi: Candida albicans and Aspergillus niger. I’ve been performing disc diffusion assays to see what plant extracts can fight which fungus. Disc diffusion assays involve placing extract-soaked discs on plates of fungus. Any anti-fungal extracts on the plates will have a “Zone of Inhibition” around them where no fungus is growing. Goldenseal has really put Candida in the danger zone: it worked better than fungicide! Garlic has proven to be a fighter against Aspergillus. You know what that means: my future is full of garlic. After the assays, any extracts that have antifungal activity will be tested on wax moth larvae. Wax moth larvae are baby moths that look like little grubs. They are often incorrectly named “worms”. I am going to infect wax moth larvae with Aspergillus and then inject them with garlic to see if having having garlic inside of them will protect the larvae from the fungus. I’ll do the same with Candida and goldenseal.
Here in the TIME sector of Brevard High School, semesters may end, but the science is never done. As the quarter has drawn to an end, final tests were being performed in HAS‘s corner of the TIME room. It had been a close competition between the fungi M. fragilis and T. asperellum the whole semester to see which species was the superior lead contamination remediator. Previously, results from the precipitate and fluorescence tests were promising but did not give an indication to which species remediated significantly more. We were sure the actual biofilter test would prove a winner, so we broke out the filter. So for each species, we got our dried fungi biomass, loaded the filter, and ran the lead water through. We used the fluorometer to measure the lead remaining and calculated the percentages remediated by each species. Unfortunately, we weren’t able to declare a significantly better bioremediator from the test… on the other hand, M. fragilis removed 97% of the lead and T. asperellum removed 96% of the lead present. Not bad stats for a prototype lead filter run on fungi.
Down here in Brevard, North Carolina science is getting real. Coming back from Thanksgiving break our minds are set on the BIG PICTURE. The Bryce, Lauren and Eliza science collaboration team are researching biofuels and the process in which enzymes are needed to produce a burnable fuel that will hopefully replace a portion of petroleum products in the future. Recently our group has been culturing several types of fungi and bacteria. These fungi and bacteria are being selected or drafted for their ability to produce enzymes valued in the biofuel process. These selected organisms will be combined to form a consortium or “team” of microorganisms which will produce enzymes in the most efficient way. Each individual organism will have a specific set of characteristics and abilities the other lacks which will hopefully provide a synergistic means of accomplishing a task. This is as if we were selecting players for a basketball team. Each player has a certain task in the operation to perform as well as possible. The Trichoderma sp. of Fungi is thought to be the “Michael Jordan” of our “players”. Trichoderma has shown abilities greater than any other fungi to produce the enzymes we are scouting for. Hopefully when our research comes to a halt we will have the dream team for producing biofuels.
This week in our project we are filtering the fungi we grew last week. We took the fungi and brought them down to Mr. Tuckey’s room for some personal time with a Buchner funnel. Each of the fungi were poured out of their containers into the funnel with filter paper and were then rinsed with 500 ml of distilled water. The container for collection at the bottom were attached to the side of a running faucet to provide a suction to speed up the process of draining the funnel. The drained fungi were then taken and put in a drying oven for use later in the heavy metal absorption tests. The collection container was then bleached and disposed of properly.
We finally managed to take some red spruce root samples a couple weeks ago. Since then, we have grown fungi from each sample on water agar plates, then potato dextrose agar. They have grown well and we have lots of interesting plates. The past few days we’ve been working on DNA extraction. We scraped a small section of fungi off each agar plate and put these samples into separate tubes. The tubes were vortexed then placed in a heat block. Today I transferred the liquid from the tubes to new tubes. This will be used for PCR and a gel electrophoresis. I am excited to see what kind of fungi we have grown!
The next step in our project will be to test the bioremediation of our fungi T. asperellum and M. fragilis. If you remember from my last blog we conducted a preliminary test by measuring the precipitate left but this is not the most accurate and its main purpose was to compare it to the other fungi we tested. This new way of measuring will allow us to use smaller amounts of metals and it will hopefully be very accurate. The florescent in presence of light will glow green and in the presence of a few heavy metals the green glow will become less strong. Using a spectrophotometer, which measures fluorescence at a certain wavelength, we can measure the intensity fo the fluorescence, therefore telling us the concentration of heavy metals present in the water.
Here in the TIME room the HAS (Hannah, Aidan, Spradlin) group has been working hard… punching fungi chips. We’re ready to begin our trial testing to determine which fungi we will be using in our in-depth heavy metal remediation tests. To narrow down to the two fungi we will use, our trial will consist of using a precipitate test to measure the amount of lead that the different species of our isolated fungi are able to absorb. Currently, we have three potential species of Trichoderma (viride, asperellum, and hamatum) and M. fragilis. We will be running the tests to narrow down out samples to one species of Trichoderma, in addition to M. fragilis. The Trichoderma species that absorbs the highest concentrations of lead will be the fungi that ‘competes’ against the M. fragilis, which was used in last year’s mycoremediation project.
So where do the fungi chips come in? Well, to control the variables in our precipitate test, we wanted uniform dried fungal biomass to work with. The question was, how to get this consistency in the material from dried sheets of fungi. The solution? A hole puncher! We spent quite some time filling weigh boats with .5g of round hole punched fungi, enough to run three trials.. But we are now ready to begin the trials. Time to see which species is the superior lead absorber.
My partner and I are studying mycorrhizal fungi. This type of fungi lives in the roots of trees and has a symbiotic relationship with the trees. It makes trees more resilient and helps them obtain phosphorous, nitrogen, water, and nutrients. Older, stronger trees can even pass necessary materials to weaker trees to help them survive using the network of mycorrhizal fungi. We are currently ppracticing our methods for our project using root samples from various baby trees and two “mother” trees. One of the first things we have to do is bleach root samples. We bleach them so that when we stain the fungi in the root we will be able to see it. Most of the samples are a yellowish color or nearly clear. Today I cut a small piece of root from the sample and looked at it under a microscope. I was able to see the cells fairly clearly. Some of the other root samples were darker and have not yet been bleached enough. Hopefully, we will be able to stain a few samples tomorrow to see if there is fungi in the roots.
In our project, we are testing the bioabsorption ability of four fungi. Biosorption is the use of biomass, in this case fungi, to absorb toxins from a solution. In our study, the toxin will be heavy metals which include lead and iron. Before we started our actual project we needed to see if our fungi would absorb the toxin while it was dead, so we used a precipitate test to determine this. We added lead to water then adding the dead fungi. Nest we let it sit and hopefully absorb the lead dissolved in the water. After this, we took a small sample of water and added Potassium Iodide because it will react with the lead, form a precipitate, and turn a bright yellow. As soon as we added the potassium iodide we could clearly see that the fungi had absorbed a majority of the lead.
The constant thought throughout this semester is “What are we doing?” Sometimes the answer is clear, while other times it takes a lot of thought. Already, Team Brella (Bryce, Eliza, and Lauren) has made a radical change to our plan. Upon further inspection of our original project proposal, we realized that it was not realistic because of our time constraints. We were so ready to begin our project, but instead we went back to the drawing board. With the help of Eliza’s perfectly organized diagrams, we came up with a new and improved plan that still works towards our goal. This is a classic situation that happens in science research. The possibilities excite and overwhelm us and we get a little dazed by them. But taking a step back and considering how realistic the project is really helps ground our ideas and makes for a great project. I can’t wait to see what happens this semester!