Quick Update on the New Column

For a quick update on my new, but simplistic, Winogradsky column, there is a photo attached here. Also, note several areas of white growth throughout the column. Most prominant is the one shown in the photo here, which seems to be clearly working on the cheese, and is also the side facing the light. These must be the fungi - or at least they are chemoorganoheterotrophs because they are feeding on organic carbon, light as well as the carbon, and using the carbon for their e- source. I partly say this because they are smack in the middle of the column and so theye strict anaerobs. Here is the photo: 

Sacrafice for the Fuel Cell!

I finally have gone and done it. I've sacrificed my favorite Winogradsky Column to the greater good - it is now a microbial fuel cell in the making. I've been carefully considering the methods used by my classmates and have finally settled on this protocol: 

http://www.emunsing.com/portfolio/portfolio_content/44_designLab/MFC_Report.pdf

I found this through the Internet geobacter site (www.geobacter.org), which has some excellent links if you scroll down and also a video clip that I hadn't noticed the first time I went to the site. 

Anyway, I used the following protocol which is to somewhat of a hybrid between the report above, my husband's engineering skills, and what I read from the other students: 

Here is a photo of some of the supplies on our kitchen counter. 

I created one very small microbial fuel cell using the my Winogradsky column from the fall semester, whose original contents were from Bear Creak. If I had mentioned before that there was no significant smell from this column, I was apparently not poking around enough. There was plenty of sulfuric smell coming from the column as I transferred it and the corresponding creak water into the MFC container. I used a 24 ounce spaghetti sauce jar. 

The anode section of the cell was made with a graphite pencil from the art store. The protocol in the research paper asks for carbon paper, but since our entire class is using graphite, I went with graphite. We also drilled all the way through the graphite and used a nut and bolt to connect the electrical wire. Because my husband, the airplane mechanic, was convinced that this would work, we then coated this area with sealant to help protect against corrosion. Since we had used a nut and bot to connect the wire, we did not use an additional connective epoxy. The anode section was then gently placed in the sediment and covered with it at the bottom of the jar. I mixed about a 1/2 teaspoon of sugar into the mud while doing this. 

The water from the column was added to the jar until it filled the jar to the surface. The Cathode was placed in immediately after. It was made in the same way as the anode. A 100 ohm resistor was placed between the wires. I tested the volts immediately after putting everything together (too soon)? I'm going to need to get guidance on the voltmeter. I am not sure what measurement everyone is using, but I read 9 micro volts initially and after 3 hours I'm down to about 3 micro volts. We'll see how this goes with time!

Here is a picture of the wires connected to the two graphite pencils: 

Here are the anode and cathode hanging off the drill while the sealant dries:

Here is a close-up photo of my poor, sacrificed Winogradsky Column after I siphoned off the water, prior to shoveling the mud into the jar: 

Here is the final product, hopefully a solid work of art and advancement for the future of mankind: 

Here is an updated look at my most active fall Winogradsky Column, which is still sitting in the window in my office. This is not a great shot, but you can make out some of the colors in the mud where there should be a mixed area of aerobic and anaerobic activity. This is especially true because I have so much water in this column. Not that much activity can be seen from possible anaerobes at the very base of the bottle, however a broad growth of clearly phototrophic organisms ( greenish, red mass) is growing on the side that always faces the window. I'm guessing that the lowest layers indicate some growth of chemoautotrophs, organisms that are using fermentation. Apparently my rubber band disintegrated and I'll have to get a new one. Also, I think it's important to note that there is no smell coming from this column, though there was a point in the past when it did have a distinctly unpleasent odor.

Spring Winogradsky Column

For the Environmental Microbiology Spring Course, I am posting a temperary blog on a simple Winogradsky Column. This one follows the very basic descriptions from this site: http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/winogradsky.html that has been recommended for the course.

I used mud from a growing puddle that has been forming right next to our pool over the last month and puddle water. I am using a glass vase that is smaller and filled greater than 2/3 of it with the mud. I used cut grasses/weeds that were growing right next to the puddle for the carbon source and cheese for the sulfer source. These were the only materials that I used in this one. We'll see what happens. I am going to try and put this column up against a light source for the duration. There is a small airspace at the top of the column.

The top of the column, being in touch with the airspace and water is already aerobic, but it is anaerobic at the bottom and mixed inbetween. If everything works correctly, and I have sulfate reducing bacteria in the mud, they will settle towards the bottom, more anaerobic section of the column and produce hydrogen sulfide gas.

Mixing the mud, grass, and cheese:

New column in glass vase. I had to work pretty hard to get all the air pockets out of this one, so bad choice overall, but I still like the way it looks.

One more of just the column before going back to the window sill:

Day 52 Observations

Winogradsky Summary for Fall Semmester:
I created 4 Winogradsky columns to see if I could show some of the various ways that microbes can survive, including in both aerobic (with oxygen) and anaerobic (without oxygen) conditions.

Column CA: In the mud in this column I placed shredded filter paper (carbon or cellulose source), magnesium sulfate (sulfur source), baking soda (sodium bicarbonate) and one crushed multivitamin pill. This column was placed in my kitchen window.

Column CB: This column was created the same as column CA, however it was enclosed in a box and kept away from light. The intent of this mixture was to provide an environment where chemosynthetic organisms could thrive without light.

Column WA: This column I mixed mud with cut grass (carbon/cellulose source) and the magnesium sulfate. Column WA was placed in the window next to column CA.

Column WB: This column was mixed the same as column WA, however it was placed in the box and kept out of the light with column CB.

After 52 days, I have seen column CA go through several stages of apparent growth. The water column at the surface went completely black for about 1 week and now has become clear again. The mud has remained almost totally black throughout, though for a few weeks there was some white areas within the upper level of the mud, closer to the water column. A thin film has remained on the surface of the mud throughout. The film was originally a whitish/greenish color and now is a thicker film that is red in color. There are red particles floating in the water column. The column has an rotten-egg like odor.

Column WA has gone through more changes. At one point the water column had taken on a foggy redish hue, but now the water is clear again. There is now a thick red film at the surface and thick red layers in the mud on the side facing the window. This column does not have a strong odor.

There has been very little change in either columns’ WB or CB. Neither column has a distinguishable smell. Both columns did go through some color change in the water column, however this was not as dark black as column WA went through. Both columns have maintained a thin film on the surface of the mud. Column WB has a thicker, redder film.

I found it very exciting to look at samples in a microscope and would recommend this part to any classroom experiment. It would be even better to have an opportunity to try to identify the microbes through isolation and gram staining.

Altogether, I think that the columns will take several more months before they can show the type of nutrient cycling and photosynthetic vs. chemosynthetic growth that the columns are known for.

Here is a photo of the surface of column WA this week:

Here are two photos of the side that has been exposed to light:

Here is a photo of the side that has been opposite the window:

In retrospect, I would have liked to use a different protocol, a contorol, and different carbon sources. I also notice that neither protocol called for a nitrogen source, so you can not follow the nitrogen cycle and it may have limited the types of bacteria that thrived in my columns. I have since found two sites that I would probably use with a class & that have a much better description of the protocol to use with students: 1. http://www.kabt.org/wp-content/uploads/2008/01/winogradsky-for-kabt-web-site.pdf
2. http://www.sciencebuddies.org/science-fair-projects/project_ideas/Geo_p038.shtml

For perspective, I also liked this site: http://steel.ced.berkeley.edu/research/hidden_ecologies/?p=31 from the Exploratorium’s Invisible Dynamics Project. This will be something to follow over time though because they’ve just got started.

Two weeks ago our lab manager helped me to use a light microscope with the computer link to take these pictures of some of my little "bugs". I only took samples from my #2 Column that is exposed to daylight. We had to use the phase contrast at 100x and add oil to the slide in order to really get a sense of what we were looking at. I also found it challenging to get a small enough sample out of the mud to be able to put it on the slide & actually see anything. Our lab manager helped me with everything & mentioned that in the first year microbiology class they are doing Winogradsky columns, but they also go through the process of isolating cell colonies on agar plates so that they have an easier time with identification. He said that for the class they use the streak-plate technique. Microbes obtained from sample aproximately 1/2 inch in mud column. Unfortunately I did not want to take up my hosts time with a staining activity (nor did I want to reveal my ignorance to the process), but that would have been even more effective in identifying what I've got here. I think if I were to do this with a class I would want to isolate some colonies and then perform gram stains to look at them.

1 micrometer =aproximately "-" on photo.
Here, I beleive you can see several living microbes taken in the picture. While looking through the microscope you could see quite a bit of movement. Most of my microbes appear to be rod shape and motile. Though there are several cocci or spherical cells.


In the lower center of the screen is a cocci shape that was pretty common with a center "nucleoid" area. It is clear that I need to purchase a microbe field guide, but even then I doubt I have the skill to satisfactorily identify these little bugs. A type of spherical cell that might be found in the conditions of my Winogradsky column could be a Micrococcus species. I pick this as a possible species because according to "A Short Guide to Some Bacteria Genera", by Harold Eddleman, Ph.D., Micrococcus species are very common in soil and dust. The colonies are also pale yellow or orange - which would match what is starting to appear in my 2nd column. They are non-motile, no spores, and aerobic which could all fit. Again, this is a sample species that might be considered, I am not going to try to identify it for real!

This is my favorite picture. The long cell shows up quite nicely and you can see the cell wall and several cellular structures inside the cell. There were several of these, but this was the best shot we got & I was unable to find it on my own (lack of patience)? For this microbe, I am going to refer to the website Microbe Wiki, http://microbewiki.kenyon.edu/index.php/Beggiatoa, for my sample species. Here I am going to highlight Beggiatoa species because of the look of the cells found on this and other sites and because the Wiki definition has this to say, "Beggiatoa is a genus of colorless, filamentous proteobacteria. With cells up to 200 microns in diameter, species of Beggiatoa are among the largest prokaryotes. They are one of the few members of the chemosynthesizers, meaning that they can synthesize carbohydrates from carbon dioxide and water using energy from inorganic compounds. Beggiatoa are found in polluted marine environments, and can be seen by the naked eye as a white filamentous mat on top of the water as a sign of environmental deterioration." I liked this description, but I don't believe I have such a mat anywhere within my column - this might rule out Beggiatoa. Also good to note that the photos on the Wiki site can be found at the Cyanobacterial Image Gallery: http://www-cyanosite.bio.purdue.edu/images/images.html.

Last, but not least, I think that the image in the middle center is another rod shaped cell with a polar flagella, there were also a number of these to be found in our samples, but this was the best shot we were able to get. I like the sample microbe that I found to highlight in this category: Rhodospirillum species. These are large spiral cells with very rapid motility and a spinning movement. According to the "Short Guide", they will grow on yeast media in the dark microaerophilically with pale white color. In the presence of light, they grow a purple or maroon color in medium anaerobically via photosynthesis. They develop huge populations in algae covered sewage ponds and can also be found in pond muds.

Currently, Column #1 has not changed much, though the water has returned to clear color, but column #2 has begun to show a variety of colors and the water has maintained it's redish hue. There is no strong smell coming from any of the columns. The columns in the dark have not developed a distinctive color change. More photos on these to be posted later.

Day 25: These photos show some of the changes that have been going on for the last few weeks. The algae in the water column seems to have multiplied in both bottles, but more noticeably in the chemosynthetic protocol column where the water appears black. The 2nd column seems to have maintained its orange tint. By following the simple diagram listed at: http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/winograd.htm I might be safe in assuming that both columns have a healthy growth of green and purple sulfur bacteria at the surface of the mud and that the 1st column has a bloom of the purple non sulfur bacteria in the water column. Possibly I have been able to promote the growth of the sulfur reducing bacteria in the first column and that might also explain the color.



Column 2: Typical Winogradsky column with cut grass & magnesium sulfate

Column 1: Chemosynthetic protocol with filter paper, magnesium sulfate, baking soda, and crushed vitamin

The two columns in the enclosed box where they are not exposed to light have shown little change, however the water column is tinted orange in both.



The 2nd column or column with grass has a thicker layer of growth at the surface of the mud and more appearance of growth just below the surface. This was not as I would have expected, as it is the chemosynthetic column that has baking soda and vitamins which were both intended to assist in microbial growth without sunlight.

I have not had a chance to check the temperature or look at any of it under a microscope yet, but I hope to be able to do so this week. I am still looking to answer why I might have so much "white" growth around the edges of the grass in the 2nd column, for which my first guess has been mold or a type of fungus.