Laboratory Exercise No. 03 Winogradsky Column

INTRODUCTION 

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 2. Anaerobic Sulfur Cycle

Aside from cyanobacteria, there are other photosynthetic prokaryotes classified in Bergey's Manual as 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

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 occurs in an anaerobic environment and 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 the anaerobic sulfur cycle (Fig 1). In this exercise an enrichment culture technique involving habitat-stimulating device called a Winogradsky column is utilized (Fig 2). Various organisms can be grown depending on their exposure to light and availability of oxygen.

Fig 3. Winogradsky Column, the simplest method of demonstrating the ecology of a number of different   

organisms

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.

OBJECTIVES

1.     To define and understand the following terms: photosynthesis, light reaction, and dark reaction.

2.     To differentiate between oxygen-producing photosynthesis and bacterial photosynthesis.

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

4.     To determine the rate of decomposition of various substrates.

METHODOLOGY

1. Stuff one handful of shredded paper into the bottom of the 1 liter graduated glass cylinder.
2. Mix marine sediment with an equal part of water and pack the column about two-thirds full with the mud mixture. Pack it to eliminate air bubbles. 
3. Add a teaspoon of nitrogen and phosphorus 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. Alternative or additional procedure can also be employed: different sediments may be used as inocula or the use different light sources (e.g., red, green, fluorescent, incandescent) and compare the colors of photosynthesizers that grow.)
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.

RESULTS AND DISCUSSION

Guide Questions

1. What was the purpose of each of the following in the Winogradsky enrichment? paper; nitrogen; phosphorous; nail?
2. Why is it necessary to eliminate air bubbles during packing?
3. Indicate the aerobic and anaerobic regions on the diagrams of your enrichment column. How can you tell?
4. Is there evidence of any non-photosynthetic growth in the Winogradsky column? Explain.
5. Why do different bacteria grow in different colored light?

         

Laboratory Exercise 3

                                                  Winogradsky Column                                                                

	Name: Date Performed Rating:

 

                 

RESULTS  

1.     Draw the predominant types of organisms present, noting the size and presence of retractile intracellular sulfur granules.

2.     Describe the characteristic changes taking place in the microbial population at different part of the column