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.

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.