List of environmental microbiology experiments

Winogradsky column

Why do different bacteria grow in different colored light?

Christine L. Case, Ed.D.
Biology Professor
Skyline College

Winogradsky Column

Environmental Microbiology
Experiments for middle and high school

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Objectives

  1. Define the following terms: photosynthesis, light reaction, and dark reaction.

  2. Differentiate between oxygen-producing photosynthesis and bacterial photosynthesis.

  3. Diagram the carbon and sulfur cycles as they occur in a Winogradsky column.

  4. Determine the rate of decomposition of various substrates.

Background

Photoautotrophs use light as a source of energy, and carbon dioxide as their chief source of carbon. The process by which photoautotrophs transform carbon dioxide into carbohydrates for use in catabolic processes is called photosynthesis. Photosynthesis can be divided into two parts: the light reaction and the dark reaction. In the light reaction, light energy is converted into chemical energy (ATP) using light-trapping pigments. Chlorophyll molecules trap light energy and provide electrons that are used to generate ATP. Carbon dioxide is reduced to a carbohydrate by the dark reaction. Carbon dioxide reduction or carbon dioxide fixation requires an electron donor and energy.

The two types of photosynthesis are classified according to the way ATP is generated and the source of electrons. Cyanobacteria, algal protists, and green plants use chlorophyll a to generate ATPs. The resulting oxygen is produced by hydrolysis (splitting water) of the electron donor, water. This photosynthetic reaction is summarized as follows:
 

Figure 1. Anaerobic sulfur cycle

In addition to cyanobacteria, there are several other photosynthetic prokaryotes. These are classified in Bergey's Manual Part I, "Phototrophic Bacteria." Most photosynthetic bacteria use bacteriochlorophylls to generate electrons for ATP synthesis and use sulfur, sulfur-containing compounds, hydrogen gas, or organic molecules as electron donors. The generalized equation for bacterial photosynthesis is:
CO2 + H2 = C6H12O6 + S
 

Figure 2. WInogradsky column.

 Winogradsky column
   

Some photosynthetic bacteria store sulfur granules in or on their cells as a result of the production of sulfide ions. The stored sulfur can be used as an electron donor in photosynthesis, resulting in the production of sulfates.

Bacterial photosynthesis differs from green plant photosynthesis in that bacterial photosynthesis:

1. Occurs in an anaerobic environment

2. Does not produce oxygen

Green photosynthetic bacteria are colored by bacteriochlorophylls although they may appear brown due to the presence of red accessory photosynthetic pigments called carotenoids. Purple photosynthetic bacteria appear purple or red because of large amounts of carotenoids. Purple bacteria also have bacteriochlorophylls.

Purple and green bacteria are involved in another biogeochemical cycle, the sulfur cycle (Figure 1). In this exercise we will enrich for bacteria involved in an anaerobic sulfur cycle.

In nature, hydrogen sulfide is produced from the reduction of sulfates in anaerobic respiration and the degradation of sulfur-containing amino acids. Sulfates can be reduced to hydrogen sulfide by five genera of sulfate-reducing bacteria (the best known of which is Desulfovibrio). Carbon dioxide used by photosynthetic bacteria is provided by the fermentation of carbohydrates in an anaerobic environment.

An enrichment culture technique involving a habitat-simulating device called a Winogradsky column (Figure 2) will be utilized in this exercise. A variety of organisms will be cultured depending on their exposure to light and the availability of oxygen.

Materials
Mud from fresh or marine water
Paper
Plant fertilizer (nitrogen and phosphorous), 1 tsp.
Iron nail or screw
Fresh or marine water
Large test tube, graduated cylinder, or clear 2-liter soda bottle
Plastic wrap
Light source
 

Procedure
  1. Stuff one handful of shredded paper into the bottom of the column.
  2. Mix mud with an equal part of water and pack a large test tube two-thirds full with the mud mixture. Pack it to eliminate air bubbles. Why?
  3. Add fertilizer and iron source to the column.
  4. Cover the column with the plastic wrap and secure with a rubber band.
  5. Place the column in front of a light source.
  6. Observe the column at weekly intervals for 4 to 6 weeks. Record the appearance of colored areas. Aerobic mud will be brownish and anaerobic mud will be black.
  7. After 4 weeks, prepare wet mounts from the purple or green patches. Record whether bacteria grew in aerobic areas; whether algae grew in anaerobic areas.
  8. Examine the paper for signs of decomposition.
 

Questions

  1. What was the purpose of each of the following in the Winogradsky enrichment? Paper; Nitrogen; Phosphorous; Nail.

  2. Indicate the aerobic and anaerobic regions on the diagrams of your enrichment column. How can you tell?

  3. Is there evidence of any nonphotosynthetic growth in the Winogradsky column? Explain.
 

Additional activities

  1. Hay and paper are the traditional starting points, however anything (e.g., styrofoam, orange peels) can be buried in the mud and examined later for the presence of bacterial colonies or other evidence of decomposition.

  2. Use different light sources (e.g., red, green, fluorescent, incandescent) and compare the colors of photosynthesizers that grow.

References

C. L. Case. and T. R. Johnson. Laboratory Experiments in Microbiology. San Francisco, CA: Benjamin/Cummings, 2004

King-Thom Chung and Christine L. Case. "Sergei Winogradsky: Founder of Soil Microbiology." SIM News 51(3):133-135, MayJune 200. View article in pdf.

Why do different bacteria grow in different colored light?

Green photosynthetic bacteria did not grow in the column illuminated with blue or green light because bacteriochlorophyll cannot absorb these colors. The green bacteria did grow in the red and yellow lights. Accessory pigments allow the purple bacteria to absorb green and blue light and grow in these columns.