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Actually Back at Wake

Friday, August 21, 2015 7:03 pm

While I never really felt like I left, I am now back to residing here at Wake Forest. This marks the “end” of my summer of research and thus, as my last post of this project, I would like to close with a brief summary of what I managed to do.

Essentially, my time with Anabaena spp. has led to two observations and one minor experiment. The first observation is the sterotypical “s-curve” of population dynamics fits to the organisms in our, lab-grown environment. The purpose of this was to fine tune further experimentation by determining in what time frame do our cultures reach full density. To test this, I used a device which scans the “optical density” of different cultures overtime. Optical density is related to how much light the culture absorbs — the denser the culture, the more light absorbed. This relates directly to the population density of a culture and thus was scientifically useful. We determined that our cultures reached density in about 5 days.

The second observation was regarding heterocyst frequency. Recall that heterocysts are differentiated cells whose sole function is to produce nitrogen. Based on the initial observation and some other, minor ones, that there may have been an indirect correlation between optical density and heterocyst frequency. It was theorized by my advisor that this may have been a genetic trade off — that the Anabaena could be dense or good at making nitrogen, but not both. Thus, we attempted to determine how heterocyst frequency varied over time and if it appeared hereditary. Samples of each culture were placed on hemocytometers — special microscope slides with grids — and heterocyst vs. vegatative cell counts were performed daily. Sadly, no conclusive results were produced.

The “experiment” was more out of scientific curiosity than need. Since my adviser’s lab is first and foremost a lab dedicated to evolution experimentation with yeast, it was posited that we might use yeast in some way in my research. It was thought that if normal yeast, who are incapable of producing their own nitrogen, could grow on the supplemented supernatant of centrifuged Anabaena cultures, then it would indicate some small amount of produced nitrogen. Thus, I spend about a week attempting to grow yeast in this manner. It was challenging because the yeast maintain a small store of nitrogen from its previous environment after transfer and cultures had to be acclimated over time to eliminate this surplus. This too proved rather inconclusive, likely due to poor design on my part.

So far, that is what I’ve accomplished. The first part of the summer was basically consumed be the development of culture production and maintenance so experimentation came late into the game. However, research is most definitely a marathon, not a sprint. For example, there is data coming out now from long term evolution experiments 20+ years in the making — tens of thousands of generations of organisms like E. coli and yeast and how they change and adapt over time. So really, I’m in no hurry.

I hope that those of you who read these few blurbs enjoyed them as much as I have enjoyed the experience behind them.

Personal Goals

Friday, August 14, 2015 6:12 pm

Hello Internet,

Still waiting on things to happen. Reagents still haven’t all been delivered. Still unsure if I’ve managed to separate my contaminant from the organisms that I want there. I seem to have burnt out the bulb on my microscope and am now just waiting. So, for this week I’ve decided to share a little about the overall goals of this project.

If my design works as intended, it will provide a new source of nitrogen products in a much cleaner fashion. Since nitrogen is so necessary (particularly in agriculture) the hope is that cutting down on these things will help us cut down on the costs of producing these compounds. This is where the real use of this system would lie. Producing nitrogen compounds cheaper would likely make fertilizer more accessible to farmers. This has different effects on the farmer depending on his status in the agricultural world. The more wealthy farmer who can already afford to buy enough fertilizer to maximize crop yields saves a few bucks. He does a small money dance and maybe passes that on to the individuals in his employ, uses it to expand his business, etc. The small farmer, however, might, for the first time, be able to buy enough fertilizer to maximize his yields. With the higher yields, his family can eat a little better and perhaps even the community as a whole can benefit from the increased food production. The smallest farmer, previously unable to afford fertilizer at all except what he can scrape together from his animals, compost, etc., uses fertilizer for the first time and notices a large boost to his normal yield. His family, for once in their life, has enough food to eat. So much, in fact, that he has extra to share with his neighbors. In a few years, the malnutrition in the farmer’s community has been cut in half.

Now, that last part is a little grandiose, but it’s not entirely far-fetched. My research has indicated that there are many place such as regions of sub-Saharan Africa where farmers of all kinds no nothing of modern fertilizer simply because of their low income. It doesn’t take much to connect these low-income, low-yield farms to hunger and malnutrition. If we can provide them with the means to make more food, there’s no telling what can happen. Without fertilizer, crop yields are likely nowhere near where they could be because of the natural growth strategies of plants when it comes to resource allocation.

Again, perhaps that too is a bit of a stretch. However it is almost guaranteed that if the system that I am designing leads to cheaper fertilizer production it will lower the operational costs of farms and increase the global food supply. I’m hoping that this will help a few people. Honestly, that’s the main goal: to help people. That and make a few bucks to go towards my building college debts. That would be nice.

Stuck in the Doldrums

Friday, August 7, 2015 3:11 pm

Well, absolutely nothing has happened since my last blog update. Isolating my cultures from there contamination is still moving but excruciatingly slowly. Things look positive however and that is most certainly worth the wait. Last I checked, my reagents for the next stage of experimentation have also not fully arrived. Thus I am simply in waiting which as it often is in the nature of scientific study. So, in lieu of something specific to mention, I decided to enlighten you all about some of the other major research occurring in my same field.

From the time that biological nitrogen fixation was discovered and we realized how integral it was to global ecology, it wasn’t long before scientists put two and two together and realized that there had to be some way to exploit this system to aid global agriculture. The largest following is an attempt to “map out” the minute genetic changes that occur to allow many of these organisms to form symbiotic relationships with plant hosts. For example, peanuts have been used for decades to revitalize farm land because Rhizobium leguminosarum, a small, nitrogen-fixing bacteria, forms a mutulistic symbiosis with the peanut plant and allows it to grow while consuming only small amounts of the soil nitrogen. This allows the soil time to naturally bring nitrogen levels up to par. Theoretically, if scientists could replicate this symbiosis with large-scale food crops such as corn, wheat or rice, it would substantially decreases the need for additional nitrogen in the form of fertilizer. In fact, if the crop yield was high enough, it might completely eliminate the need. So why am I headed in a different direction? The intricacies of that system are extremely elusive and, in my opinion, many years away from being truly understood. This is mostly because many of these organisms must differentiate into a specialized cell dedicated to nitrogen fixation. These systems are typically found to be extremely sensitive — so much so that we typically cannot observe them under traditional techniques. Thus, I think my system is a more realistic under taking until modern science can get this system figured out.

Other scientists are trying to use combinations of either the bacteria in whole or just the nitrogenase system into new, biologically active fertilizers. This system, along with other biological systems to enhance phosphorous and potassium levels, hope to simply produce better fertilizer. This is a phenomenal idea if it could be done but, a simple chemical product as intended out of my system would simply be easier to use.

Overall, I have seen no indication that other researches are pursuing something similar to the system I am trying to produce. While I highly doubt that is true, it is comforting in the fact that I might be able to make this system into something that could provide some financial gain for myself and others in addition to its inherit ecological benefits. I hope that holds true.

Swimmers Part II

Tuesday, July 28, 2015 2:16 am

Well, after returning from vacation I was both pleased and not-so-pleased with the results of the current methods of attempting to salvage my poor, contaminated cultures. The antibiotic that we treated the cultures with had, in fact, the opposite of our intended affect. It appears to have killed off some portion of the Anabaena and caused the swimmers to multiply exponentially. This does seem to indicate that the swimmers are feeding off of the Anabaena in the cultures as adding an antibiotic shouldn’t provide a food source by itself. This may help us in determining what these buggers are and, in turn, maybe a chemical method of dealing with them.

We also explored some methods of physically separating the Anabaena from the contaminating species. Essentially, liquid cultures can be “streaked” onto solid agar plates in such a way that multiple concentrations of anything within the liquid cultures appear on the plate. From there, individual colonies can be isolated from the others within the culture and used to inoculate fresh cultures. Hopefully, this method will have more positive results than the antibiotic.

The only other thing of interest is that I will soon be starting a new assay with a more biochemical focus. We’ve finally managed to order reagents for an assay that allows for the detection of nitrogen by converting ammonia into a bright blue product. With the help of a spectrophotometer, the amount of colored particles like in this reaction can be measured and thus indirectly measuring the amount of ammonia in solution. Hopefully, this assay will help us determine the amount of nitrogen being produced and left in solution by our Anabaena cultures. That is … if they’re not horribly contaminated. We’ll see how it goes I suppose.

Swimmers

Thursday, July 16, 2015 2:29 am

Today we had a potentially small catastrophe. It appears that the cultures that I am working with are contaminated with a small organism I have affectionately dubbed “swimmers.” Under the microscope, the little buggers look like little black ovals. When they move, they seem to oscillate similar to how flagella (cellular tails some organisms use for locomotion such as human sperm cells) do so in a movement referred to as a “power stroke.” I’m currently attempting to find ways in which to destroy these organisms without hurting the Anabaena residing in the culture. Hopefully, tomorrow will show that one or more of these attempts are successful. While having to trash these cultures isn’t a huge tragedy, it would most certainly be a set back. This is because these populations have been propagating and evolving over the course of about two months. The only remaining cultures then are the ancestors of the cultures that are now contaminated. Thus, any beneficial mutations that may have occurred since this project began will be lost. So disastrous? No, not really. Rather frustrating? Most definitely. Luckily, I’ve there are ways to prevent this from occurring in the future. It’s a common practice to store various cultures on ice by placing them in liquid gas cooled freezers. These freezers are so cold (for example, liquid CO2 freezers in the WFU Bio building maintain a temperature of about -84 C) that cultures can be held for long periods of time in essentially a form of stasis. For some time, usually months or years, the cultures can be thawed and grown without any major problems. So, implementing a freezing regiment will help prevent such large loss in the case of future contamination. We’ll see how it goes.

Rinse and Repeat

Monday, June 29, 2015 9:16 pm

You first hear about the scientific method in middle school: question, hypothesis, experiment, conclusion, repeat. However, in most of our minds, the “repeat” part gets left out. Sure they say it’s important, but no 6th grader thinks anything after the bubbling paper mache volcano is very interesting. One of the lessons I’ve learned over the last several weeks is how much repeating in good science their actually is. For example, it occurred more than once that I would run a seemingly simple experiment only to be looking at the data to realize some fatal flaw: some small but consequential variable had not been accounted for or my technique wasn’t quite right. Even with all the knowledge we have in today’s society, the process of trial and error is often forefront in many a scientific endeavor. Even looking into the future, with all due conscience, weeks of “playing with the science” in an effort to determine how best to go about answering a certain question is inevitable. This is not a complaint, but a realization and a new-found appreciation for the common researcher. Not to mention, a shocking realization of how far humanity has progressed over the last century despite this tedious process. Almost mind-boggling really.

Stand Back … I’m Doing Science Part 2

Friday, June 19, 2015 4:22 pm

Hello Again,

So to bring you all out of the pit of suspense that I put you in last week. Essentially the idea is that there is a family of organisms known as diazotrophs. These organisms do the same thing for themselves that requires a giant, hotter than Hades, pressurized vat. Thus, my research focuses around how to develop a new method of producing nitrogen surrounding these organisms. Now the possibilities are endless: for example, many researchers are trying to develop new biofertilizers using derivatives from these organisms. Thus, currently my research is focused on analyzing and studying these organisms: learning what they do and how they do it. I am hoping it will come to some method to which I can exploit this unique ability for the betterment of the world. We’l see how it goes.

Stand Back … I’m Doing Science

Thursday, June 11, 2015 10:21 pm

The word “internship” is a bit off in my situation. This summer I am spending a large portion of my time working in the lab of Dr. Clifford Zeyl of the WFU Department of Biology on a very special project I am happy to call my own. I am in the process of attempting to re-design a system fundamental to human life — though, you yourself have probably never heard about it. The industrial scale production of nitrogen compounds has become a large market due to its key role in the production of agricultural fertilizer. The importance of fertilizer is highly underrated — without it, crop yields would ultimately not be large enough to sustain our rapidly increasing population. However, the process in which we make nitrogen compounds for fertilizer and other such needs is a costly process. Atmospheric nitrogen (N2) is pumped into a large chamber along with hydrogen gas (H2) and is put under extreme heat and pressure (between 400-600 degrees Celsius and 400-600 atmospheres of pressure) in the presence of a metal catalyst. This system produces ammonia (NH3) which is substantially more user-friendly that N2, whose bond strength is high that it is extremely inert. The ammonia is then used either directly or as a precurser to a large variety of useful substances such as fertilizer. While this system is extremely useful, the extreme conditions are costly to produce and maintain. The hope is that my project can produce a new system that will not require such conditions. “How’s that?” you might ask. That is a question for next week’s blog (insert joke about cliff hangers here).

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