Growing Chemosynthetic Bacteria

Growing Chemosynthetic Bacteria

Introduction: Scientists once thought that sunlight was source of energy for all life
and that photosynthesis was the only way to make food (fix carbon, make glucose
~ sugar). It is now known that reduced chemicals (molecules that can donate
electrons) from hydrothermal vents provide chemosynthetic energy for some life
forms. High temperatures and high concentrations of dissolved minerals in seawater
form “reduced” compounds such as hydrogen sulfide. Using complex processes
called biochemical pathways, bacteria oxidize hydrogen sulfide (forcing H2S to give
up electrons) and use the liberated energy carrying electrons to produce
carbohydrates (i.e., stored chemical energy). Unlike photosynthesis,
chemosynthesis requires no light and can occur at the extreme temperatures and
high pressures of the deep ocean. The chemosynthetic food web supports dense
populations of uniquely adapted organisms.

Chemosynthetic bacteria (Archaebacteria) may be one of the oldest life forms on
Earth. In 1880, long before the extreme bacterial habitats like deep-sea
hydrothermal vents or dark cave ecosystems were discovered and studied, a
Russian scientist named Sergei Winogradsky discovered the bacteria Beggiatoa.
Theses bacteria metabolize hydrogen sulfide (H2S) to produce the energy for
making carbohydrates (glucose, sugars). He made this discovery by making a
habitat in the laboratory later called the Winogradsky column --developed long
before hydrothermal vent or cave ecosystems were discovered, Beggiatoa is among
the bacteria found in the deep-sea hydrothermal vent environment, but it is not the
only bacteria to take advantage of this chemosynthetic process as you will soon
discover.
In this activity, your class will construct two “Winogradsky Column”, grow and
observe the chemosynthesis and succession of bacterial colonies: one column will
be placed in the sunlight, the other in a dark place. The Winogradsky Column will
enrich and isolate certain organisms involved the cycling of sulfur and nitrogen in
their mini-ecosystem “column” environment. This activity will allow you to observe
over time how living things use chemosynthesis to make their food and the
ecological process of succession (when one species becomes more dominate in a
community of living things while other species become less dominate because of
changes in the local living conditions).
Adapted from: http://www.bigelow.org/foodweb/chain4.html

Adapted from Orange County Marine Institute / San Juan Institute Activity Series AND "Visit to an Ocean
Planet" CD-ROM, Copyright 1998, California Institute of Technology and its licenses

Learning Objectives:
• Some organisms cannot draw energy from the sun and must find other
energy sources to live
• Both photosynthesis and chemosynthesis are means of producing
carbohydrates
• Photosynthetic organisms use light as their energy source; chemosynthetic
organisms use chemicals
• As organisms thrive in a given environment, their by-products create a new
environment where new species can succeed

Before the experiment begins, give students a tutorial on what to look for in their
cylinders. In the first week, students should see green-colored algae in the well-lit
column. Then, over a period of six weeks, at least five different bacteria may grow in
succession in both columns. It is difficult to know exactly what bacteria are actually
growing in the columns. The first species may be the anaerobic (i.e., living in the
absence of oxygen) bacterium Clostridium; this heterotroph (i.e., requires organic
material for food) would use the straw or filter paper as a carbon source to produce
food. Another bacterium, Desulfovibrio, may use the waste of Clostridium as its source
of carbon and CaSO4 as an energy source. Desulfovibrio may produce the hydrogen
sulfide required by the rest of the ecosystem. Three other bacteria -- Beggiatoa (white
or yellow), Chlorobium (green), and Chromatium (purple and violet) -- use hydrogen
sulfide as part or all or their energy source to make food; because they also require
oxygen, you will find these bacteria near the surface of the sediments. After formation
of purple and green bacterial patches, black spots of hydrogen sulfide will likely
appear. Hydrogen sulfide will be identifiable by its distinctive odor.
Assignment / Extension Activities:

• For at least six weeks, examine the columns weekly and look for signs of
bacterial growth. You may wish to use a safety light (flashlight covered with red
cellophane) to examine the columns being grown in the dark. Record your
observations.
• Bacteria that use light as their major energy source with some hydrogen
sulfide are heterotrophic. Bacteria that use hydrogen sulfide for energy in the
absence of light are chemotrophic. Are the bacteria in your well-lit and darkened
columns similar?
• Based on your results and the description above, can you distinguish which
bacteria are heterotropic? Chemotrophic?
• Discuss how the by-products created by some types of bacteria were used by

other types of bacteria that then became the dominant species. (This process is
called "succession.") Can you name other examples of "species succession"?
• (OPTIONAL) At the end of the third week take samples for microscopic wet
mounts observation from the following locations: (1) surface layers of water, (2)
surface layers of the mud, (3) colored layer from the mud. Try using a pipette and
be careful not to disturb the column. Observe the wet mounts under high-power

Adapted from: http://www.bigelow.org/foodweb/chain4.html

Adapted from Orange County Marine Institute / San Juan Institute Activity Series AND "Visit to an Ocean
Planet" CD-ROM, Copyright 1998, California Institute of Technology and its licenses

magnification, looking for cell shapes that would indicate the types of organisms
present.
Materials For Each Group:
1. Two 2-liter bottles or 500 ml graduated cylinders,
2. Enough black mud to fill these cylinders 2/3rds full,
3. CaSO4 (Plaster of Paris: found in any hardware store),
4. Beakers/buckets/pails for mixing,
5. Stirring rods,
6. Organic straw (200-350 ml by volume if using 2-liter bottles or 50-75 ml by
volume if using 500 ml graduated cylinders)
7. 3 liters of pond water (or seawater or swamp water),
8. 8 grams baking soda if using 2 liter bottles or 4 grams baking soda if using 500ml
graduated cylinders,
9. Multivitamin pills and something with which to crush them,
10. A Cap for the 2-liter bottles, plastic wrap if using 500ml graduated cylinders
11. Rubber bands, Light source that can stay on for at least six weeks,
12. Tape and markers for labeling columns,
13. Flashlight with red cellophane on lighted end (for later use)
14. Divide the class into two groups. Each group of students will set up one of two
identical columns. One will be kept in the dark and the other will be placed under
a light source. Obtain mud from a local lake, river, or bay or estuary. If it is not
completely black let the mud sit for a while in a jar to blacken.
Procedure:
1. Add about eight grams of CaSO4 (for if using 500ml graduated) to enough mud
to fill one 2-liter bottle about 2/3rds full (if using a graduated cylinder to a depth of
about 8.0 cm. Dump the mixture into a mixing container and stir it thoroughly with
the stirring rod.
2. Measure the volume of straw and cut it up into little pieces. Place the straw in the
mixing container with the mud and mix gently. (It may help to add some
pond/swamp/sea water here to ease stirring.)
3. Add to the mixture about 0.2 grams of baking soda and 4 crushed vitamin pill
(add on if using a graduated cylinder). Stir again to make sure all the air bubbles are
gone.
4. Transfer the mixture back to the 2-liter bottle or cylinder using a funnel and a
push rod and add pond or seawater so that the mud is covered with at least 8
centimeters (3.2 inches) of water
5. Set the cylinder aside for 30 minutes to settle.
6. After 30 minutes, if more than two centimeters of water have pooled at the top,
pour off all but one centimeter (0.4 inches). If there is less than one centimeter of
pooled water on top, add pond/swamp/sea water.
7. Repeat steps (1) through (6) for the other graduated cylinder.
8. Label the 2-liter bottles or cylinders with the class’s period number.
9. Place one graduated 2-liter bottles or cylinder in a darkened area where it will not
be disturbed for at least six weeks. Place the second 2-liter cylinder under the light
source. (You may wish to store both set-ups in the same area with one cylinder in a
box. This will help to keep both in similar conditions.)
Adapted from: http://www.bigelow.org/foodweb/chain4.html

Adapted from Orange County Marine Institute / San Juan Institute Activity Series AND "Visit to an Ocean
Planet" CD-ROM, Copyright 1998, California Institute of Technology and its licenses

10. Record smell, color, number of layers of mud, or any other observations.
Adapted from: http://www.bigelow.org/foodweb/chain4.html

Adapted from Orange County Marine Institute / San Juan Institute Activity Series AND "Visit to an Ocean
Planet" CD-ROM, Copyright 1998, California Institute of Technology and its licenses