Sergei Winogradsky was the pioneer in the investigation of microbial autotrophy (and microbial ecology in general) in the late 1800's and early 1900's, and was a strong proponent of examining freshly-isolated organisms rather than domesticated laboratory strains. One of the methods he developed for the study of microbial nutrient cycling in the environment is what is now known as the Windogradsky column. These can be set up in an amazing variety of ways to study sulfur, nitrogen, carbon, phosphorus, or other nutrients, most often cycling between the upper aerobic zone and the lower anaerobic zone.

In our case, we let microbes generate sulfide at the bottom of the column. Cellulose fermentation slowly releases sugar for use by anaerobic organisms. Sulfate (added as calcium sulfate) then serves as an electron acceptor for sulfate reducers, generating sulfide. Carbonate is available for autotrophic growth and as a pH buffer. We use relatively insoluable calcium salts so that they don't create an overly salty environment. Metal (typically iron) sulfides create a black color at the bottom of the column. Sulfide diffuses upward into the column, and oxygen diffuses downward from the surface. Sulfur oxidizing organisms consume both where they meet, resulting in stable counterbalancing sulfide and oxygen gradients. This allows organisms of any oxygen or redox requirements to grow. In our case, we're particularly interested in looking for conspicuous sulfur-cycling organisms and photosynthesizers.

• mud or sediment - the richer & blacker the better
• rain or pond water
• 1 foot length of thin plastic tubing (these come from fluorescent light covers)
• #9 rubber stopper
• cellulose powder, or filter paper confetti
• calcium carbonate
• calcium sulfate
• parafilm

1. Firmly stopper one end of your platic tube with a #9 stopper. Tape this stopper in place and use this for your label.
   
2. add 1/2 spoonful each of cellulose powder, calcium carbonate and calcium sulfate to the bottom of the tube.
   
3. Pack the column 1/2 to 3/4 full with the mud, being careful not to trap large air pockets.
   
4. Top off the column with pond or rain water.
   
5. Clean off the outside of the column and cover the top with parafilm.
   
6. Place in the lighted 30C incubator.
7. Incubate for weeks to months. Top off with water as required. Here is what a typical column looks like at weeks 0, 1 and 2:
   
8. Sample the liquid and top of the sediment periodically, looking microscopically for interesting organisms. If your liquid phase has more than one layer, sample each layer and the interface between layers. If you have colonies growing on the glass in the liquid phase, scrap some off & have a look microscopically. Also check to meniscus - sometimes there are interesting things at the air:water interface.
9. When the column is well-developed, pour out the water and carefully slice the tube open on two sides. Gently scrape and wash out the sediment. Use a metal spatula to scrap colored biofilms from the wall of the cylinder or from the sediment. Examine microscopically.

Check weekly. Make detailed observations (a labeled drawing might be best) on the appearance of the column, from bottom to top and from the light to the dark sides, especially regarding stratification, color, and obvious signs of life.

Key to potential observations:

• Aerobic colors
  • green - eukaryal algae or cyanobacteria
  • red/brown - cyanobacteria or thiobacilli
  • red/purple - purple non-sulfur Bacteria
  • white - sulfur oxidizing Bacteria
• Anaerobic colors
  • red/purple - purple sulfur Bacteria
  • green - green sulfur Bacteria
  • black - sulfate reducers
• Gas ...
  • ... in the water column is probably O2 from oxygenic photosynthesis
  • ... in the aerobic zone is probably CO2 from respiration
  • ... in the anaerobic zone is probably CH4 from methanogenesis
• Tracks in the upper layers of the sediment are formed by " worms"
• Small specks swimming in the water column are crustaceans, e.g. Daphnia & Cyclops.

There will be a lot of variation in the columns made using various muds and waters. Here are some example photographs:

This column contained a lot of sulfide and very sandy sediment that allowed the sulfide to diffuse easily into the bulk of the column, and as a result is anaerobic throughout. Notice the purple sulfur Bacteria (Chromatium, in this case) at the top of the sediment, and green sulfur Bacteria below them. The water column is full of sulfur oxidizing Bacteria, such as Beggiatoa.

This column, on the other hand, contains less sulfide and compact, clay sediment that only allows slow release of sulfide into the column. The column is aerobic in the water column and the top of the sediment. There is a dense mat of filamentous cyanobacteria at the top of the sediment, many colonies of purple sulfur Bacteria deep in the sediment, and numerous cyanobacterial colonies on the surface of the glass in the water column. Bubbles of gas generated and trapped in the deepest part of the column are probably methane.

Notice the fine layering of the top of the sediment, and the green and purple blotches on the glass in the sediment layer. White growth near the top of the sediment are sulfur oxidizers, and mark the boundry between aerobic and anaerobic zones.

This column had no sulfide at all and is poor in organics; as a result it is completely aerobic. It is rich in cyanobacteria and eukaryotic algae, and generates lots of gas (probably oxygen). Because of the lack of toxic sulfide, the water column is rich in protists and crustaceans, and there is a plant and a small snail as well.