Algae Microbiome: The Hidden World

Pressley Wilson, University of South Carolina Aiken

The Approach: In my previous post, I discussed (1) the importance of an organism’s microbiome in relation to its health and (2) the importance of algae in marine ecosystems due to their ability in producing oxygen, removing nitrogen and phosphorus from water, and exchanging inorganic carbon.

Considering the importance of these two variables, this summer I am researching the relationship between the algae microbiome and algae species in One’ula Beach, Honolulu, Hawai’i.  

DNA extraction in progress. Photo credit: Dr. Heather Fullerton.

The objectives of my research project are:

  • Identify relationship between algae microbiome and algae species
  • Identify relationship between the microbiome with algae’s morphology

Prediction

This study will predict there is a variation in the microbiome between algae species, due to the different species characteristics, such as calcification.

Sample Collection

The five algal species of different morphologies were hand-sampled by Dr. Heather Spalding at the intertidal region of One’ula Beach. The algae samples were rinsed with artificial saltwater to remove dirt and loosely associated bacteria. After cleaning, each species were placed into (1) a micro-centrifuge tube with 0.5 mL of RNA-later or (2) a 15 milliliter conical tube with 1.5 mL RNA-later. The RNA-later is a DNA preservation agent, was used to stabilize the algae DNA. The samples were stored at 4°C overnight. This overnight incubation allowed the RNA-later to penetrate the bacterial and algal cells to the DNA. After this incubation all tubes were frozen at -20°C and shipped overnight on dry ice to the College of Charleston, South Carolina, where they were stored in  -80°C freezer until DNA extraction.

Sample Analysis a

A completed gel electrophoresis.

A MoBio Fast DNA Spin Kit was used to extract the DNA from the algal samples. This DNA is then tested by PCR to determine if bacteria are present on the algae. To determine the number of bacteria present, qPCR is used. This molecular biology technique is used to quantify specific genes in a sample.

The PCR samples will be analyzed using gel electrophoresis, a molecular biology procedure that uses an electrical current to separate the components of the sample DNA by size. The qPCR data will be compared to a known DNA standard to determine the number of bacteria in our samples and calculations will be performed using excel.

Acknowledgements

I would like to thank Dr. Heather Fullerton for her guidance and support with this project and Dr. Heather Spalding for her sample collection. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899. 

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Meddling with Mysterious Macroalgae

Pressley Wilson, University of South Carolina Aiken

The problem: If you have ever been in the ocean, you have probably come across a piece of seaweed, which is a type of macroalgae. Macroalgae are simply algae that can be seen without a microscope. These organisms undergo photosynthesis, produce carbon, and can reduce the levels of phosphates and nitrates in water (Champagne et al. 2015).

Although algae are one of the most important parts of marine ecosystems, the algae microbiome (the bacteria that live in and around algae) is highly unknown and further research is needed to uncover this critical macroalgae information. Is there a relationship between bacteria and algal species? Is there a relationship between the algae’s physical features and the bacteria? Or is the microbiome the same throughout the algae, regardless of variation in species and physical features?

In order to answer these questions, I am conducting a research project this summer looking at the bacteria that are associated with intertidal macroalgae from One’ula Beach, Hawai’i.

Intertidal region of One’ula Beach, Hawaii

The five species below were chosen from the intertidal region of One’ula: Asparagopsis taxiformis (1), Avrainvillea sp (2), Halimeda discoidea (3), Padina sanctae-crucis (4), and Dictyota sandviscensis (5). These species were chosen because they range from red, brown, and green algae; have varying physical features; and all currently grow on the intertidal region of One’ula Beach.

Macroalgae from One’ula Beach, Hawaii (Photo credit: Dr. Heather Spalding)

Asparagopsis, Avrainvillea, and Dictyota are uncalcified, Halimeda is calcified, and Padina is lightly calcified. Asparagopsis has fluffy upright filaments, Avrainvillea and Padina have a fan-shaped thallus, Halimeda has flattened segments, and Dictyota has dichotomous branches. All species are native to One’ula Beach, Hawaii, except Avrainvillea which is an invasive species, meaning it is not native to the area. These species represent a diverse array of brown algae (Padina and Dictoyta), green algae (Avrainvillea and Halimeda) and one red alga (Asparagopsis).

This research will lead to a better understanding of algae, which could lead to a better understanding of all photosynthetic marine organisms. Furthermore, this research will be used as preliminary results for Dr. Heather Spalding’s work in the Northwestern Hawaiian Islands determining if there is a relationship between spatial patterns and the algae microbiome, beginning in August.

Acknowledgements

I would like to thank Dr. Heather Fullerton for her guidance and support with this project and Dr. Heather Spalding for her sample collection. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899. 

References

Champagne P, Hall G, Liu X, Wallace J, Yin Z. 2015. Determination of Algae and Macrophyte Species Distribution in Three Wastewater Stabilization Ponds Using Metagenomics Analysis. MDPI – Water. 7(7): 3225-3242.

Uncovering Seasonal Changes in the Algae Our Oceans Depend On

Emily Spiegel, Bryn Mawr College

As described in my previous posts, this study focused on a polar diatom, F. cylindrus.  Despite the harsh temperatures of its habitat, this diatom is awesomely productive. It can form blooms under sea ice so thick, it looks like grass! Marine organisms feed on these blooms, which contributes to productivity of the entire ecosystem.

Because the poles are situated at the ends of the Earth, they are subject to constant changes in light availability, from continuous light to continuous darkness. How are photosynthetic organisms like F. cylindrus able to adapt to this stressful change? Their ability to produce biomass is dependent on light levels: too much and these cells can be overwhelmed, too little and there may not be enough to balance against the costs of respiration.

I found that in the low light exposure of polar autumn (6h light: 18h darkness), F. cylindrus begins to reproduce sexually, instead of asexually. This was found through analysis of RNA expression, which is an indicator for how much a certain gene is being transcribed into proteins to do work within the cell. Sexual reproduction leaves behind a trace in the RNA, based on the particular genes involved. As opposed to the primary form of diatom reproduction (asexual), sexual reproduction conserves resources and produces fewer cells. So the population does not grow to the same extent as populations reproducing asexually, but it’s also able to survive in stressful and changing conditions better than asexual populations.

Interestingly, stress can also reduce the ability of F. cylindrus to remove carbon dioxide from the atmosphere, in a process known as carbon fixation. This shift could have major implications for how well the polar oceans remove CO2 from the atmosphere at different times of year. Could autumnal months in the poles show dramatically decreased carbon fixation rates? What would such a pattern mean for current global carbon models? Further research must be conducted at the poles themselves to determine whether this relationship exists in nature, and how it is affecting carbon flux within the polar oceans.

This research was conducted in the lab of Dr. Peter Lee from the College of Charleston at the Hollings Marine Laboratory in collaboration with the Medical University of South Carolina. Many thanks to all members of the lab, particularly Nicole Schanke, MSc.

Some Dramatic Microorganisms and Targeted Genetic Analysis

Emily Spiegel, Bryn Mawr College

IMG_4143

Genetic analysis has become the name of the game in many fields of biological research. Genes encode proteins, and in biology, proteins are king. Proteins guide biological pathways throughout the entire organism, so if you can track the genes, you can understand how the animal functions. Advances in technology like CRISPR, RNA sequencing, and PCR have improved the accessibility and accuracy of high-level genetic analysis in laboratories across the world. Some scientists utilize this technology to study the entire genome of an organism, while others attempt to understand the response of specific genes to various environmental factors or other external influences. This summer, I’m conducting an experiment focused on the latter. I’ll be studying how the polar algae species, Fragilariopsis cylindrus (affectionately known as Frag) copes with environmental stress by reproducing sexually. To do so, I’ll use targeted RNA sequencing to track genes related to sexual reproduction.

In order to understand how a Frag, responds to environmental stresses, you need a lot of algae. I reared nearly 100 liters of this algae in different artificial conditions. These conditions varied by two factors: photoperiod (the length of day and night), and nutrient levels. If you missed my previous post, “Stressing Out My Algae,” you should check it out for more details on the background for this experiment. We suspect that in conditions of stressful light energy (24 hours of continuous light), Frag will respond by reproducing sexually as opposed to its normal asexual mode of reproduction. This could possibly be a mechanism to rid itself of excess energy in times of stress, since sexual reproduction is more energetically expensive than asexual reproduction. By reproducing sexually, Frag may improve its chances of survival against this stress. Compounded with this is our hypothesis on nutrient deprivation. Previous experiments have shown that when a major nutrient, nitrogen, is limited, the algae cannot grow at full capacity and sexual reproduction is inhibited. We predict that when the stress of nitrogen limitation is combined with the stress of high light energy, we’ll see a reduction in the algae’s ability to survive in the stressful conditions due to the inhibition of sexual reproduction. So if we stress out the Frag enough and take away their ability to have sex, they’ll probably die. They’re some very dramatic microorganisms.

IMG_4131

24 bottles of algae were grown in six different experimental conditions varied by length of light exposure and nutrient levels. Algae was reared in 4-liter bottles filled with seawater.

So we grew our Frag, four bottles per six experimental conditions. Every day for eight days we extracted biomass from the bottle. From this sample we could test chlorophyll levels and cell counts, both of which give us a good idea of how well the algae in that bottle are growing in their conditions. We also took samples to be used for RNA extraction. Remember how genes encode proteins and proteins are king? Well before you can have your protein product, you need RNA. You’ve probably heard of DNA, which is the double stranded genetic cookbook. RNA is its single stranded offspring, which is then used as a the direct template to make proteins. A lot of genetic analysis therefore looks at RNA instead of DNA in order to understand how genes are being transcribed for protein production. We’re currently working on extracting the RNA from the original biomass sample and then running that pure RNA through a specialized machine called Nanostring. This is extremely targeted analysis, as Nanostring focuses in on the specific RNA we’re most interested in. In this case, we’re interested in RNA which is encoded from genes related to sexual reproduction. Using Nanostring will tell us how active the genes for sexual reproduction are in each bottle, which we can analyze to derive any correlation between our environmental stress factors and sexual reproduction.

If our hypothesis is correct, then we’ll see the greatest expression of sexual reproduction genes in the conditions of high light energy (24 hours of continuous light). We’d expect to also see low growth performance in nitrogen limited populations, indicated by low cell counts and chlorophyll levels. In these populations we predict we’ll see little if any expression of genes related to sexual reproduction. By the end, we’ll hopefully have a clearer picture of how phytoplankton like Frag deal with environmental stress.

Funding for this project is provided by the National Science Foundation in collaboration with the College of Charleston Grice Marine Laboratory and the National Oceanic and Atmospheric Administration. Acknowledgements to the entire lab of Dr. Ditullio and Dr. Lee in the Hollings Marine Laboratory facility.