Gas Chromatography for Fatty Acid Analysis

Jack McAlhany, Wofford College

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In applying to College of Charleston REU at Grice Marine Laboratory, I assumed I would be researching the well-being of a local fishery or the biomechanics of an organism in response to different variables, but this was not the case. My research to this point has not focused on a living organism, but rather tiny portions of blood plasma that we vaporize into a gas to determine the basic constituents of the blood. A solid understanding of chemical structures, or a wild imagination, is necessary for this research because unlike working with live samples, our desired product is not visible. To give you a better understanding, the fatty acids we are detecting have between 8 and 24 carbon atoms while a 70 kg human has approximately 7X10^26 carbon atoms (Kross, B.).

In order to detect such tiny fat molecules I am using a gas chromatography with a flame ionization detector (GC-FID). This instrument converts a liquid sample to a gas that then travels through a 100 meter column with an inert gas carrier. The time at which certain samples exit the column is reproducible, so the retention time in the column is a way to determine which fatty acids are in a blood plasma sample (E.T.S. Laboratories).

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Figure 1: In a GC-FID, a sample (red) is vaporized in a vaporizing column (blue) and an inert gas carrier is the “liquid” phase that carries the vaporized fatty acid methyl ester (FAME) through a 100 meter column (gold). This column has a stationary phase that interacts differently with each FAME, causing the FAME’s to exit the column at different times. The detector responds to compounds that form ions when combusted in the hydrogen-air flame. This response is converted to electrical signals and produce a gas chromatogram we can analyze (E.T.S. Laboratories).

These retention times are represented on a gas chromatogram, which consists of peaks of varying sizes (Figure 2). Each fatty acid interacts in a unique, but constant, way with the stationary phase of the column, so fatty acid peaks are in the same order and have the relatively same retention time every trial. This constant retention time allows us to determine the fatty acid composition of a sample. Not only can we find the blood plasma fatty acid composition, but by analyzing the area under each peak, we can determine the concentration of each fatty acid in the blood. The concentration of each fatty acid or relationships in these concentrations hopefully will provide a biomarker that we can test in the field for the fatty acid disease, pansteatitis.

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Figure 2: Gas Chromatogram of 19 fatty acids. Each peak is labeled with its corresponding fatty acid (Christie W. 2011).

Acknowledgements

I would like to thank the College of Charleston for this internship, the National Science Foundation for funding, and Dr. John Bowden for his guidance as a mentor.

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Works Cited

Christie, W. 2011. Gas Chromatography and Lipids. The Oily Press” Chapter 10.http://lipidlibrary.aocs.org/GC_lipid/10_misc/index.htm

E.T.S. Laboratories https://www.etslabs.com/analysis.aspx?id=$GCP

Kross, B. Questions and Answers. Jefferson Lab. http://education.jlab.org/qa/mathatom_04.html.

Grac Attack!

Aaron Baumgardner, The University of Akron

The only thing more pervasive than the constant thoughts of, conversations about, and stress from Gracilaria vermiculophylla in Dr. Erik Sotka’s lab is the invasion of this red alga that is occurring along the coasts of North America and Europe (Sotka, et al. 2013). After only a few short weeks in Charleston, I have seen how prevalent and successful this seaweed is. The success of G. vermiculophylla along the southeastern coasts of the United States is due in part to the established mutualism between it and the decorator worm Diapatra cuprea. This mutualism provides a secure site for the seaweed to grow (Kollars, Byers, and Sotka unpublished manuscript), but it does not explain the success of G. vermiculophylla to thrive in environmental conditions that differ from its native Japanese range.

G. vermiculophylla colonizes a mudflat in Charleston Harbor by clinging to tube-building decorator worms. Credit, Erik Sotka

G. vermiculophylla colonizes a mudflat in Charleston Harbor by clinging to tube-building decorator worms. Credit, Erik Sotka.

Researching G. vermiculophylla can help us understand how aquatic invasions occur. Do introduced populations evolve novel characteristics or do they simply benefit from the phenotypic plasticity of their source populations? To answer this question, it is necessary to test plasticity in response to varying environmental conditions on native and non-native populations (Huang, et al. 2015). Since G. vermiculophylla has spread outside its latitudinal range and into high salinity environments (Kollars, et al. 2015), I will be testing the plasticity of native and non-native G. vermiculophylla populations to a range of temperatures and salinities. Photosynthetic efficiency will be measured using a PAM fluorometer to provide a more objective way to quantify stress (Rasher and Hay 2010).

I would like to thank the College of Charleston for this internship opportunity, Dr. Erik Sotka for mentoring me on my project, and the National Science Foundation for funding REU programs.

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References:

Huang, Q. Q., et al. (2015). Stress relief may promote the evolution of greater phenotypic plasticity in exotic invasive species: a hypothesis. Ecology and Evolution 5(6), 1169-1177.

Kollars, N. M., Byers, J. E.,  & Sotka, E. E. (unpublished manuscript). Invasive décor: a native decorator worms forms a novel mutualism with a non-native seaweed.

Kollars, N. M., et al. (2015, in review). Development and characterization of microsatellite loci for the haploid-diploid red seaweed Gracilaria vermiculophylla. PeerJ.

Rasher, D. B., & Hay, M. E. (2010). Chemically rich seaweeds poison corals when not controlled by herbivores. PNAS 107(21), 9683-9688.

Sotka, E. E., et al. (2013). Detecting genetic adaptation during marine invasions. Grant proposal to the National Science Foundation.

A day in the Shrimp Lab

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Alessandra Jimenez, Whitworth University

Have you ever wondered what it’s like to be a lab researcher who works with live animals? Through this internship, I am experiencing this firsthand in Hollings Marine Laboratory, along with all the responsibilities involved!

A normal workday in the life of a “shrimp intern” is like this: A big part of it is animal care and maintenance. It starts in the morning with a daily visit to the wet lab, where approximately 80 brown shrimp juveniles are kept in four large tanks with circulating water. After feeding them a round of commercial shrimp pellets, I test the salinity of the water in each tank using a refractometer to make sure that each tank has a certain salinity value: 30 parts per thousand, to be exact. I use dechlorinated freshwater and seawater to adjust this value if needed. Besides salinity, I also need to watch out for harmful levels of ammonia (it’s a part of shrimp waste!), nitrates, etc. In usual circumstances, I conduct a water change (replacing old water with new) once a week in order to dilute these chemicals. For the past couple of weeks, however, I have been conducting water changes daily in order to keep ammonia levels neutral in three tanks. Ah, the life of a caretaker of tons of baby shrimp!

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Wet lab. @AlessandraJimenez

Besides animal husbandry, I work on my experiment involving the effects of injection of bacteria on tail flipping (Want to learn more about what I’m doing? click here). I have two shrimp at a time in separate, well-aerated tanks, and they are both from the same treatment group. Shrimp are randomly assigned to one of four treatment groups. These treatment groups are designated according to the treatment type (injection of bacteria or saline) and according to the amount of time between the moment of injection and the tail-flipping procedure (4 or 24 hours). I randomly select two shrimps from the wet lab, weigh them, and keep them in the two experimental tanks overnight so they can get used to the new environment, temperature, etc. The next day, I take each shrimp out of the tank momentarily and quickly inject them with bacteria, or a saline buffer if they are part of the control group. Then, I give them 4 or 24 hours (depending on group type) to rest before conducting the actual tail-flipping experiment. Using a stir-rod (basically, a straight stick), I poke the shrimp lightly to induce tail-flipping, and count how many flips they perform before fatigue. The number of flips here is called ‘initial activity’. Then, I give them 20 minutes to recover in the tank before tail-flipping them again. The number of flips this time is called ‘recovery activity’.

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Experimental tanks @AlessandraJimenez

Why tail-flip them twice? Well, we hypothesize that recovery activity will be impaired in bacteria-injected shrimp versus the controls, while initial activity would probably not be. This is based on how recovery from tail-flipping activities involves aerobic (or oxygen-fueled) metabolism. Since bacteria accumulate in the gills of shrimp and block oxygen uptake (want to learn more? click here), it would make sense that recovery activity would be reduced. Stay tuned for results later on!

Works Cited:

Gruschczyk, B., Kamp, G., 1990. The shift from glycogenolysis to glycogen resynthesis after escape swimming: studies on the abdominal muscle of the shrimp, Crangon crangon. J Comp Physiol B, 753-760.

Scholnick, D. A., Burnett, K. G., & Burnett, L. E. (2006). Impact of exposure to bacteria on metabolism in the penaeid shrimp Litopenaeus vannamei. Biological Bulletin, 211(1), 44-49.

Many thanks to College of Charleston for hosting my project, Dr. Karen Burnett and Hollings Marine Laboratory for guidance and work space, and NSF for funding the REU program.

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Humans and gators and chickens, oh my!

Jimena B. Pérez-Viscasillas, University of Puerto Rico at Mayaguez

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When I first applied to this Marine Biology REU, in a lab that works mostly with alligators, at a Marine Science Campus right by the Charleston shore, I never thought I’d end up working with chickens. Yes, you read correctly: chickens, of the Chick-Fill-A and Kentucky Fried sort. I was surprised too, naturally, but it turns out the reason behind it is actually pretty important.

A couple of years ago, a group of scientists noticed some alligator populations in Florida weren’t doing too well. Their fertility levels were decreasing and a lower percentage of the eggs laid were hatching. Upon further study, evidence pointed towards a likely culprit: anthropogenic chemical contaminants in the environment. These contaminants were negatively affecting the gators’ hormone production and, in turn, their reproductive systems.

What do these gators have to do with chickens, though? Perhaps more importantly, what do they have to do with us? Let’s review some basic bio…

Figure 1: Vertebrate phylogenetic tree. Amniotes are organisms which have adapted to terrestrial reproduction. This group includes birds, reptiles, and mammals. (Graphic taken from: UCL)

There are some terrestrial animals which lay eggs (like chickens and gators) and some that carry their young in the womb, inside the placenta (like us). Both types of organisms, collectively called amniotes, have much of the same tissues surrounding their embryos during development. This shared characteristic means that we may be able to study some egg-laying animals to better understand our own reproductive systems.

Figure 2: A chick embryo and membrane. The membrane I’m going to be studying is that which lines the inside of the shell. Its called the chorioallantoic membrane, and it allows gas and waste exchange between the embryo and the environment. (Taken from Angiogenesis Laboratory Amsterdam)

Before we can use these organisms’ tissues as models of our own, however, we have to make sure we understand how they function. This is where I (and the chickens) come in. This summer, I’m going to be measuring how (and if), at different stages of development, the egg membrane of chickens produces hormones called prostaglandins. Prostaglandins play a major role in the immune system, as well as the body’s general regulation and reproduction. This preliminary research would help us better understand these sentinel species and allow us to later assess how their endocrine, immune and reproductive systems are being compromised by environmental pollutants. If we know how chemical contaminants in the environment are having negative effects on their reproduction, what might it tell us about how they’re affecting our health and reproduction?

To learn more about my project, check back for further posts!

Acknowledgements

This research, conducted at Dr. Louis Guillete’s MUSC Laboratory, is made possible thanks to funding from NSF and the College of Charleston. Further equipment and facilities are provided by the Hollings Marine Laboratory.

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References:

Bellairs, Ruth & Osmond, Mark. The Atlas of Chick Development.  San Diego, California: Elsevier Academic Press, 2005. Print.

Guillette LJ Jr. “The evolution of viviparity in amniote vertebrates—new insights, new questions.” J Zool  223 (1991): 521–526. Web. 10 June 2015.

Guillette LJ Jr. “The evolution of viviparity in lizards.” Bioscience 43 (1993): 742–751. Print.

Kalinski P. “Regulation of Immune Responses by Prostaglandin E2.” J Immunol 188 (2012):21-28. Web. 10 June 2015.

Kluge AG. Chordate Structure and Function. New York: Macmillan Publishing Co., Inc.; 1977. Print.

Milnes MR, Guillette LJ Jr. “Alligator Tales: New Lessons about Environmental Contaminants from a Sentinel Species.” BioScience 58.11 (2008): 1027-1036. Web. 15 June 2015.  doi:10.1641/B581106

Making Renewable Energy An Even Cheaper Alternative!

Yoel Cortes-Pena, Georgia Institute of Technology

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Fig 1. Picture of me

I’m Yoel Cortes-Pena, a chemical engineering senior student at Georgia Tech and future scientist and entrepreneur.  My research interests lie in renewable energy and environmental sustainability. Additionally, although I am an engineering student during the day, I am also part of Hip-Hop culture at night. My hobbies include dancing, beatboxing and rapping. Here is a link to my channel. 

Through this blog, I want to share with you my research experience as part of the Fort Johnson Undergraduate Summer Research Program. When I received the acceptance letter, I was surprised and happy that I would be working with Dr. Harold May in Microbial Electrosynthesis. This new technology uses microbes to fix carbon dioxide and electrons from an electrode to produce fuels and highly valued chemicals such as hydrogen, methane and acetate.

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Fig 2.Picture of Microbial Electrosynthesis Reactor. The graphite rod on the left is the cathode (electron donor) and the rod on the right is the anode (electron acceptor). The left side of the reactor is being sparged with CO2. The microbes, located on the left side of the reactor, are fixing the CO2 and producing hydrogen (visible bubbles) and acetate (dissolved in solution).

One of the many applications of microbial electrosynthesis includes the storage of energy without contributing to carbon emissions. Solar, wind and other renewable energy forms output a variable amount of energy that tends to exceed public demand, especially during off-peak hours. Consequently, this surplus electricity becomes stranded energy that cannot be used. Microbial Electrosynthesis can utilize this excess or stranded energy and store it in fuel, valorizing the use of renewable energy technology.

Acknowledgements

Dr. Harold May’s Enviromental Microbiology lab is affiliated to the Medical University of South Carolina (MUSC).This project is possible thanks to funding from the NSF College of Charleston Summer REU program and the Grice Marine Laboratory. Lab space and facilities are provided by the Hollings Marine Laboratory.

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New Philadelphia to Charleston

Aaron Baumgardner, The University of Akron

Coming from the landlocked small town of New Philadelphia in the Midwest, I feel like I’m dreaming when I realize I’m spending my summer researching in Charleston, SC. I’m thankful for the opportunity that my mentor, the College of Charleston, and the National Science Foundation has given me to learn and grow in my scientific ability.

However, I do not believe I would be where I am today if it weren’t for my Aunt Jane. She is the only member of my family with a background in science, and even though she is hundreds of miles away at UPenn, she is always an email or phone call away. She has always shown an interest in my academics and will always be there for any advice I may ask. She has helped me develop my professionalism and offered insight on which graduate schools are worth going to.   Because of her, I can finally realize it’s not a dream. It’s reality that I’m spending my summer in Charleston, SC. It’s because I’ve worked hard in school and reached out for opportunities for me to mature as scientist. And I owe her so much for pushing me to succeed.

Thank you Aunt Jane!

The real beauty of coral reefs

Nina Sarmiento, Binghamton University

The beauty of a coral reef is undeniable. Over four thousand species of fish, 800 species of coral, invertebrates, and large macro fauna coming together in one place is sure to create a thrilling visual experience. You might be surprised to learn that these remarkable places filled with twenty five percent of marine life, constitute less than one percent of the ocean floor.1 But you don’t have to be lucky enough to travel to a coral reef to fully appreciate its beauty. The real value of reefs comes from their unsuspecting roles in sustaining life as we know it.

photo cred: fmap.ca

photo cred: fmap.ca

Fish from approximately half of our global fisheries, at one point spent a part of their life in coral reefs.2 The unique habitat hard corals provide is perfect for spawning and juvenile life for many species, which may later end up in other parts of the ocean. Fishermen make their livelihood from these reefs, harvesting an average of fifteen tons of seafood annually per square kilometer.3

As for people living on our tropical coastlines, reefs play a crucial role in protecting life on land. It is in the beauty of the long braches of Copra palmata, among other corals, that dangerous storms and waves are softened. Corals roughness and their shallow locale dissipate wave energy, and we have a natural barrier that safeguards our homes.4

Acropora palmata – “Elkhorn coral” Photo cred: coral.aims.gov.au

Acropora palmata – “Elkhorn coral”
Photo cred: coral.aims.gov.au

The importance and intrigue of coral reefs has led to studying many of the organisms and interactions there, leading to new understandings of many aspects of organism biology and evolution. Additionally research has uncovered new medicine from extracting compounds unique species have, giving reefs an importance in future medical interests.

The paradox is that, of all the reasons why we appreciate coral reefs, it is we, the human species that are not having a good effect on them. In fact we are seeing reef decline in many parts of the world because of our actions.5

This summer I am delving into studying one of the possible reasons for this decline; a chemical threat to coral that may not be obvious at first, but could have significant implications on their ability to survive and reproduce.

Stay tuned to hear about my project and the amazing opportunity I have to be a part of the effort to preserve these beautiful communities.

References:

1 Spalding MD, Ravilious C, Green EP. 2001. United Nations Environment Programme, World Conservation Monitoring Centre. World Atlas of Coral Reefs. University California Press: Berkley. 416.

2 US Coral Reef Task force. 2000. The National Action Plan to Conserve Coral Reefs. Washington DC: US Environmental Protection Agency. 34.

3 Ceasr H. 1996. Economic Analysis of Indonesian Coral Reefs. Washington DC: The World Bank.

4 Lowe JR, Falter JL, Bandet MD, Pawlak G, Atkinson MJ, Momismith SG, Koseff JR. 2005. Spectral wave dissipation over a barrier reef. Journal of Geophysical research. 10: C04001.

5 Nystrom M, Folke C, Moberg F. 2015. Coral reef disturbance and resilience in a human-dominated environment.

Funding for my research comes from the National Science Foundation in partner with The College of Charleston and the National Oceanic and Atmospheric Administration

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