The Summer of Algae

At the start of my work this summer, the intent was for me to work with cleaning algal samples with hydrogen peroxide and determining a method for thinly spacing the cleaned frustules for imaging with a scanning electron microscope (SEM).  I was able to take some pictures of the frustules using the Phenom SEM at the Applied Research Center (ARC) but the samples were too congested with debris and other frustules for the images to be of any use other than my learning.  Because the frustules are very small with complex, intricate features, the SEM can distort the images of these objects.  So to get around this, lots of images need to be taken at different times to avoid the phenomenon known as charging up as I discussed in an earlier post.  For this to happen, I must be able to return to the same frustule many times, hard to do when there are thousands of similar looking ones and sediments.  However, because of constraints with supplies, the majority of my time was focused on constructing a craft that would contain screens for algae growth.  This required using the machine shop to create features for this craft to hold as well as raise these screens out of the water.  We are working in the York River, so this makes for a challenging environment.  Because of the salt water, many commonly used materials cannot be used as they will corrode, such as steel (stainless is great though).  Our main challenge this summer was creating a pulley system for raising the screens out of the water.  This might not sound like such of an issue but these screens will have algae growing on them (hopefully).  Algae is mostly water, so its decently heavy when wet.  Our first design for a cross-plate to hold the screens was a quarter inch thick aluminum plate.  This significantly bowed under the weight of screens and a small amount of growth the first time we tried to raise them.  So we switched to a type of reinforced aluminum bar which held up much better.  The junction between the poles that supported the pulley system and the crossbar holding the screens was a problem area.  The crossbar would twist and bind on the pole so that it would only move up a foot or two if we were lucky in the beginning.  We redesigned this region to reduce the amount of contact between the poles and the crossbar to a bare minimum. It works ok now, the weight is still an issue though.  On the very last day of our research experience, we were able to take the craft out to its mooring location in the York River.  It had previously been sitting in the boat basin at VIMS.  However, the combination of strong current and choppy waters made it so that we were not able to reinstall the screens which had been removed for transport.

Hydrogen Peroxide Fun

In the closing weeks of my research experience, Dr. Cooke tasked me with finding a procedure for determining the actual percentage of hydrogen peroxide that was in a sample.  The hydrogen peroxide was used in cleaning to prepare an algal sample for imaging under a scanning electron microscope.  Very small amounts of the hydrogen peroxide (30%) are actually used in the cleaning and so I had been using a smaller sample separate from the larger container.  This container went through multiple cycles of heating and cooling, so it was natural to ask whether the amount of hydrogen peroxide is comparable to the apparent starting value of 30% by volume or if the sample had degraded significantly after the heating cycles.

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Algae Progress

Another aspect of my work this summer is obtaining calorimetric values for samples of mainly algae and hydroids.  I am using a Parr 6400 calorimeter, which shows the amount of heat energy contained within a sample in terms of calories per gram.  The top performing algal samples thus far are a type of red algae with an average output of 2052.09 cal/g, while the hydroid samples are our overall top performer with an average output of 3075.88 cal/g.  The algal samples have trouble igniting by themselves in the calorimeter and so a spiking substance, solid benzoic acid, is added as an accelerant.  The calorimeter that I am using is able to account for the heat produced by the benzoic acid and subtract that amount so that the “gross heat” figure given by the calorimeter concluding a test is the heat energy released by the sample.  I would also like to test samples of Enteromorpha before the end of this session.  The other side of this project has been growing algae on our craft and designing/implementing a pulley system to raise the screens on which the algae is growing out of the water for collection and maintenance.  At the moment this craft is still in the boat basin at VIMS but will be deployed before this session is over.

Imaging Algae

I have taken my first images of the algae using the Phenom scanning electron microscope (SEM) at the Applied Research Center (ARC).  These pictures have shown that there was too much algae on the stub.  I need to be able to image a particular frustule and then return to that same individual multiple times after modifying it or examining it in another piece of equipment. In addition, I must be sure that the patterns seen on the frustule are indeed the actual structure, not a result of charging up.  Charging up is when electrons build up in a region on the surface of whatever is being imaged.  This causes the paths of the imaging electrons to be distorted and so the resulting image is skewed.  This usually shows up as static-like lines across the image or just very bright sections.  However, the effect of charging up can be much more localized so as to distort minute features, especially as the magnification is increased.  From these stubs, I did find that previously dried samples cannot be used unless they are washed and filtered significantly more.  The algae are just simply not separated from the sediments and other material.  For the same reason, centrifugation can’t be used because it creates a solid mass rather than separating.  The stubs that had algal samples that had been continuously suspended produced much better images.  These samples were cleaned using room temperature and 40C 30% hydrogen peroxide.  These cooler temperatures revealed a sample that had a larger proportion of intact frustules than previous cleanings.  These cooler temperatures allowed for new algal structures to be viewed but were not terribly effective at removing the sediments and other material that doesn’t interest us.

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more algae

I have been able to identify the two major genera of algae that largely dominate the samples collected from this location on the York River.  I have also been exploring methods of cleaning these samples using both heated and room temperature 30% hydrogen peroxide. The samples have been both dried and continually suspended samples with better results coming from the previously suspended samples.  Under a simple compound light microscope, the algae appeared to have most if not all organic matter cleaned from their frustules when using the suspended samples.  However, part of my goal of the project is to establish a temperature at which the cleaning is optimized, meaning that most if not all of the surrounding organic matter is gone with the frustules staying intact.  I am stuck in this respect as we have ordered a digital thermometer that is on back order at the moment.  I have also learned the basics of using a calorimeter and spiking samples of algae and hydrozoa to derive the amount of available energy they contain.  The goal at the moment is to fully assemble the portion of our craft on which the algae is to grow and be collected from.  Going forward, I need to prepare and test samples using the calorimeter, develop a protocol with more parameters for cleaning the frustules, maintain the craft to make sure algae is growing optimally, and image samples of algae using the SEM microscopes and HIROX microscope at the Applied Research Center.