ENVIRONMENTAL MICROBIOLOGY - M. I. Chernyavskaya - 2016

TOPIC 1. WINOGRADSKY COLUMN

Studying The Diversity of microorganisms is impossible without isolating pure cultures, as this provides reliable data on The properties of specific microorganisms and their modes of interaction. In natural environments, pure cultures of microorganisms are rarely found; therefore, the first step in their isolation is obtaining enrichment cultures. The primary objective here is to create optimal conditions for the growth of a specific species or group of microorganisms relative to others.

To obtain an enrichment culture and subsequently isolate photoautotrophic, chemolithotrophic, and chemoheterotrophic microorganisms, the Winogradsky Column is a convenient tool. It also serves as a model that illustrates the spatial distribution of various groups of microorganisms within a body of Water, primarily those associated with carbon and sulfur METABOLISM. A crucial pattern revealed by observing The Development of the Winogradsky column is The Emergence of a vertical gradient of redox conditions (ranging from anaerobic at the bottom of the column to aerobic at the top) and the creation of ecological niches for the development of different groups of Bacteria. Typically, in the first few days after Setting up the column, anaerobic decomposition of organic matter begins within the sludge mass, catalyzed by a group of hydrolytic microorganisms and primary anaerobes (fermenters). Fermentation products (Н₂, organic acids, alcohols) are utilized by secondary anaerobes, including sulfate-reducing bacteria. The metabolic products of the latter (sulfide and СО₂) diffuse into the medium and serve as substrates for the growth of anoxygenic phototrophic (Purple and green) and chemolithotrophic (thionic, colorless sulfur) bacteria. Anoxygenic phototrophic bacteria develop in the anaerobic zone of the sediment and water, forming colored layers or patches on the side of the column facing the light. In the upper part of the column, cyanobacteria and Algae appear, releasing O₂ in the presence of light. Thionic and filamentous colorless sulfur bacteria can develop under conditions where H₂S diffusing from below and O₂ coming from above are simultaneously present. At various time periods and in different Zones of the column, conditions are established for the development of numerous groups of microorganisms that make up the aquatic ecosystem (Fig. 1). The completeness of their detection depends on the column's incubation time and the thoroughness of observations.

Class="center">Fig. 1. Diagram of the distribution of various groups of microorganisms in a Winogradsky column

Review Questions

1. Define THE CONCEPT OF "environmental microbiology".

2. List the main research directions in the field of environmental microbiology.

3. What types of interactions can occur between microorganisms?

4. What types of interactions can occur between microorganisms and macroorganisms?

5. What is The Significance of enrichment cultures in microbiological practice?

6. Which groups of microorganisms does the Winogradsky column allow to study?

Laboratory Work 1. Obtaining an enrichment culture of photoautotrophic and chemolithotrophic microorganisms

Objective: to set up a Winogradsky column and observe its development over 6 — 7 weeks.

Materials and equipment: Glass columns (cylinders) at least 30 cm high and 8 cm in diameter (volume — 500 — 1000 mL), pond sludge or mud (250 — 500 g, depending on the cylinder volume), calcium carbonate (СаСO₃), calcium sulfate (CaSO₄), shredded newspaper or filter paper, a bowl or container for the sludge, a spoon or spatula for mixing, distilled water, a piece of aluminum foil, a plastic lid or parafilm, a light source (a window or fluorescent lamp).

Procedure

1. Collect sludge/mud from a water body (the sludge must be fresh, so sampling must be carried out on the day the column is set up).

2. Remove debris from the sludge/mud sample.

3. Transfer 125 — 250 g of sludge/mud into the mixing bowl (container).

4. Add 5 g of СаСO₃ and 5 g of CaSO₄.

5. If the sample is not moist enough, add a little water.

6. Mix thoroughly.

7. Add 10 g of shredded filter paper or newspaper and mix again.

8. Place the resulting mass into the column (the column should be filled to approximately 1/4 — 1/3 of its capacity) and pack it down tightly to remove any air pockets.

9. Transfer the remaining sludge/mud into the column (to a level of approximately 5 cm from the top) and pack it down again.

10. Add water (it should cover the sludge/mud by 2 — 3 cm).

11. Wait for the water to settle (30 min). There should be 2 — 3 cm of headspace left above the water in the column. If there is too much water, remove the excess; if there is too little, add more until the required level is reached.

12. Seal the column tightly with Parafilm and then with foil.

13. Incubate the column for 6 — 7 weeks at room Temperature in indirect sunlight (on a windowsill).

14. Check the column every week to observe its development.

15. Record the observations in a laboratory notebook.