Tag Archives: #APBioGradsky

What’s that in my Seagrass?

This past weekend we took Bethany Dixon (@MsBethDixon)’s class of AP Bio students out to Bodega Marine Laboratory to make winogradsky columns from seagrass sediments. For more information about what we were doing, why we did it and what the heck a winogradsky column is see Jenna’s previous posts, AP Bio Winogradskies and AP Bio Winogradskies Pt 2. As part of the experience, the students had to opportunity to wade out into a seagrass meadow to collect samples and thus the mysteries begin! Follow along on twitter with #APBioGradsky!

UNSOLVED MYSTERIES:

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What is that I see in my seagrass roots? What is that is that sticky white stuff? Fungi? Underwater spiderweb?

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When placed underwater this strange organism looks like a wiggly worm brain – what the heck is it?? Zombie slug?

AP Bio Winogradsky Recipes

This past weekend we took Bethany Dixon’s class of AP Bio students from Western Sierra Collegiate Academy out to Bodega Marine Laboratory to make winogradsky columns from seagrass sediments. For more information about what we were doing, why we did it and what the heck a winogradsky column is see Jenna’s previous posts, AP Bio Winogradskies and AP Bio Winogradskies Pt 2. For more winogradsky goodness see: Adventures with Winogradskies, Further Adventures with Winogradskies, Even More Adventures with Winogradskies and On the Road Again: Adventures with Winogradskies. Keep up to date with these columns by following along on twitter with #APBioGradsky!

How to Make a Winogradsky: We gave the AP Bio class a standard winogradsky recipe and then five “experimental” recipes. These recipes are described below.

Standard (10x Recipe)  
cellulose  1 g
sodium sulfate  1 g
ammonium chloride  0.1 g
calcium carbonate  0.1 g
potassium phosphate  0.1 g
diatomaceous earth  300g

This is the standard recipe which we are using for our “control” columns.

Potassium Nitrate (10x Recipe)  
cellulose  1 g
sodium sulfate  1 g
ammonium chloride  0.1 g
calcium carbonate  0.1 g
potassium phosphate  0.1 g
diatomaceous earth  300g
potassium nitrate  0.1 g

This recipe adds potassium nitrate to the standard recipe. By adding potassium nitrate, we hope to encourage denitrification (the process by which microbes take in nitrate and produce N2). By encouraging denitrification, we hope to enrich for microbes potentially involved in the nitrogen cycle.
Ammonium Acetate (10x Recipe)  
cellulose  1 g
sodium sulfate  1 g
ammonium chloride  0.1 g
calcium carbonate  0.1 g
potassium phosphate  0.1 g
diatomaceous earth  300g
ammonium acetate  0.1 g

This recipe adds ammonium acetate to the standard recipe. By adding ammonium acetate, we hope to encourage nitrification (the process by which microbes take in ammonia and produce nitrite). By encouraging nitrification, we hope to enrich for microbes potentially involved in the nitrogen cycle. Also ammonium acetate provides an additional carbon source for the microbes and should help encourage microbial growth.

Iron(III) Phosphate (dihydrate) (10x Recipe)  
cellulose  1 g
sodium sulfate  1 g
Iron(III) Phosphate (dihydrate)  0.1 g
calcium carbonate  0.1 g
potassium phosphate  0.1 g
diatomaceous earth  300g

This recipe replaces ammonium chloride from the standard recipe with Iron(III) phosphate (dihydrate). Iron is a necessary co-factor for nitrogen fixation and both iron and phosphorus have been posited to co-limit nitrogen fixation in the ocean. By adding iron(III) phosphate, we hope to encourage nitrogen fixation (the process by which microbes take in N2 and produce ammonia). By encouraging nitrogen fixation, we hope to enrich for microbes potentially involved in the nitrogen cycle.

Seagrass Roots (10x Recipe)  
Seagrass roots  1g
sodium sulfate  1 g
ammonium chloride  0.1 g
calcium carbonate  0.1 g
potassium phosphate  0.1 g
diatomaceous earth  300g

This recipe replaces cellulose from the standard recipe with seagrass roots. Since we are trying to culture seagrass associated microbes, we thought that it would be interesting to use seagrass roots as the carbon source for the winogradsky columns. We hope that this will result in an enrichment of microbes that from symbiotic relations with seagrass roots.

Elemental Sulfur (10x Recipe)  
cellulose  1 g
sodium sulfate  1 g
ammonium chloride  0.1 g
calcium carbonate  0.1 g
potassium phosphate  0.1 g
diatomaceous earth  300g
elemental sulfur  0.1g

This recipe adds elemental sulfur to the standard recipe. By adding elemental sulfur, we hope to enrich for microbes involved in the sulfur cycle.

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Collecting seagrass and sediment!

Inoculation: We had the students prepare their recipes on their first day and then inoculate them on their second day. We inoculated them with seagrass sediment and water collected from Bodega Bay by the AP Bio class. We mixed the sediment and water collected vigorously and let the sediment settle. The water was then used to inoculate the columns.

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Inoculating our winogradskies!

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The finished columns! #APBioGradsky

AP Bio Winogradskies (Pt. 2)

Even with a few days distance from the (hopefully) inaugural field trip with the AP Bio class from Rocklin Collegiate Academy, I’m still shocked by how smoothly everything went. On Friday, we set up the Winogradsky columns (see post here.) On Saturday morning, were were so fortunate to be at the Bodega Marine Lab on the day when UC Davis Biological Sciences freshman are bussed out to the BML for a tour and some proselytizing for marine biology as a field of study. Because there was already staff on hand for the freshman tours, we were offered the opportunity to have our class go on the same tour. After the tour and an inspirational overview, given by Dr. Eric Sanford, of ongoing research at BML, we had lunch and then killed time at the dorms while waiting for the tide to go down.

I’d borrowed 3 pairs of waders for the students who were adventurous enough to get into the chilly water to collect seagrass plants and sediment. However, after polling the students, I realized that almost everyone was pretty excited about putting on the waders, so I grabbed a bunch more. At 2:30, low tide, we went to Westside Park to hunt for seagrass beds. From the shore, it was pretty easy to spot the dark patches in the water that I assumed were them. So, we walked across the mudflat for about 100 yards and then just a few feet into the water, they were right in the thick of them!

We gave them spades and buckets and they dug right in. The buckets were filled up about halfway with sediment and plants, and topped off with seawater. After about an hour, we had to drag them away, they loved looking at all of the crazy marine invertebrates and eggs that were attached to the leaves. Back at the lab, we stirred the buckets, let the sediment settle a little, and then loaded up the columns. They’re now in the hands of WSCA’s AP Bio class, and we’re really looking forward to watching them develop!

AP Bio Winogradskies

For the Seagrass Microbiome project, one thing we’d like to do is to build a culture/reference genome collection for microbes found in association with seagrasses. For now, we are just working on this for Zostera marina, both because it is our primary experimental model organism and because we have easy year-round access to it at Bodega Bay.

However, while we hope to do more of it in the future, the Eisen lab isn’t currently set up to isolate the types of organisms that we think are likely to play an important role in seagrass adaptation to marine sediments, e.g., sulfur-oxidizing microbes (which require anaerobic conditions to grow). So, for now, we are using an enrichment tool known as a Winogradsky column to grow lots of sulfur oxidizers (and other things, but we’ll focus on sulfur oxidation here.)

In Winogradsky columns, sulfur and oxygen gradients cause microbes to stratify themselves according to their ability to utilize the available electron donors and acceptors. These microbial layers are visible to the eye, making Winogradsky columns a great classroom tool for teaching students about microbial physiology, biogeochemistry, ecological niches, succession, and much more.

Bethany Dixon is an AP Biology teacher at Western Sierra Collegiate Academy, a charter school in Rocklin, California. She has been building Winogradsky columns in her classroom for a couple of years now. We have teamed up to have her students build Winogradsky columns that have been inoculated with seagrass bed sediment. One thing we know is that we will be able to enrich for seagrass-associated microbes with a variety of metabolic capabilities. But, we don’t know much more than that. For example, how many different species will be oxidizing sulfur in these columns? Will the same microbial species perform sulfur oxidation in every column that’s made from the same sediment? What will happen if we add a bunch of elemental sulfur to the column? Will that change the makeup of the sulfur oxidizing community in the columns? Will it affect the shape of the sulfur gradient? Will it affect how quickly the layers form in the columns? Will it kill the column?

So, we arranged for Bethany’s AP Bio class to come to UC Davis’ Bodega Marine Lab (BML) to collect some sediment and inoculate some Winogradsky columns. Typically, Winogradsky columns are built by mixing sediment with a carbon source (like newspaper) and a sulfur source (like egg yolk), topping it off with water, and then sealing it. I got the idea of using diatomaceous earth as a physical matrix and laboratory chemicals for the carbon source (cellulose) and sulfur source (sodium sulfate) from Tom Benoit at McMurry University. We just make a slurry of the sediment and water, let the big particles settle, and then add the water to the columns.

This is really cool because we can control and systematically vary the chemical composition of each column. So, with the AP Bio class, we have defined 5 experimental “recipes” and each student is making two columns, with one experimental and one standard recipe. We’ll discuss the five experimental recipes in a follow-up post.

Today, the students arrived at the BML. We spent the afternoon weighing out and mixing chemicals, and loading our 50ml conical tube columns. Tomorrow, at low tide, we’ll head over to a seagrass bed, load up a bucket with sediment, and haul it back to the lab to inoculate the columns!

You can follow our progress here, or in real-time on Twitter with the hashtag #APBioGradksy

Visiting the experimental seagrass tanks. One of the experimental recipes involves substituting macerated seagrass roots for cellulose. Everyone visited the experimental tanks to grab some seagrass roots for those. They would have spent hours here, checking out the different tanks, watching the sea stars, urchins, and sea hares.
Visiting the experimental seagrass tanks.
One of the experimental recipes involves substituting macerated seagrass roots for cellulose. Everyone visited the experimental tanks to grab some seagrass roots for those. They would have spent hours here, checking out the different tanks, watching the sea stars, urchins, and sea hares.
Ecosystem engineers. Back in the lab, a classroom provided by the BML, five teams weighed out the chemicals for their experimental columns, and loaded them up.
Ecosystem engineers. Back in the lab, a classroom provided by the BML, five teams weighed out the chemicals for their experimental columns, and loaded them up.