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Contents:
Topic Template
Introduction:
Absorption by Chlorophylls of Photosynthetic Organisms
Lab Protocol: Making a Winogradsky Column
Extend: Investigating Electrochemical Potential
Lab Protocol:
Chlorophyll Extraction and Absorption from Unknown Samples
Photos of Kit Components
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Topic Template Topic Associated Curriculum Associated Content Materials Required 5E Learning Cycle • Engagement • Exploration • Explanation • Elaboration • Evaluation Related NGSS Standards Background/Why Lab 1 Lab 2 Absorption by Chlorophylls of Photosynthetic Organisms Solar Energy Photosynthesis, Biodiversity, Ecology, Electrochemistry, Energy Transfers, Light as EM Waves • A Handful of spinach leaves • Graduated cylinder • 10 – 15 mL Acetone • Shredded newspaper • Filter Paper • Calcium sulfate/egg yolk • Funnel • Calcium carbonate/powdered chalk • Test tube • Wooden dowel • Mortar and Pestle •
Plastic wrap • Black Light and Dark room • Rubber band • Rock-­‐free mud • Aluminum foil • Pond water • Describe a pond you’ve seen before. What colors do you remember, what sounds, what insects, etc. • Construct an apparatus to grow various species of microorganisms which are photosynthetic. • What role do photosynthetic organisms play in an ecosystem? • How would changes to the population of photosynthetic organisms affect the population of other organisms? • Why do photosynthetic organisms tend to grow close to the surface of a water system? MSLS1-­‐4. Use argument based on empirical evidence and scientific reasoning to support an explanation for how characteristic animal behaviors and specialized plant structures affect the probability of successful reproduction of animals and plants respectively. MSLS2-­‐1. Analyze and interpret data to provide evidence for the effects of resource availability on organisms and populations of organisms in an ecosystem. MSLS2-­‐4. Construct an argument supported by empirical evidence that changes to physical or biological components of an ecosystem affect populations. Some microorganisms also contain chlorophyll molecules which can absorb different spectra of light. This energy is used to drive photosynthesis, but in the absence of this pathway the energy can be seen by the naked eye. Making a Winogradsky Column (Growing a host of organisms, including various photosynthetic organisms) Chlorophyll Extraction and Absorption from Known Samples (separating and comparing the spectra of photosynthetic organisms versus various known organisms) 3 Introduction: Absorption by Chlorophylls of Photosynthetic Organisms
WHY?
Photosynthesis takes place in plants, algae and bacteria. Photosynthesis occurs when the
energy from light is used to drive electron transfer. While the details differ in various types of
organisms, the basic chemical mechanism is essentially the same in all organisms that do
chlorophyll-based photosynthesis.
Engage:
Describe a pond you’ve seen before. What colors do you remember? What sounds? What
insects? What else do you remember?
Explore:
How can you construct an apparatus to grow a various species of microorganisms, which are
photosynthetic?
Background:
In this lab, you will grow various species of microorganisms, which are photosynthetic and then
characterize the absorption spectrum for a variety of pigments extracted from known
photosynthetic bacteria as well as from unknown organisms extracted from the Winogradsky
column.
A Winogradsky column is a vertical pond environment used to study diverse microbial
populations. The column is named after Sergei Winogradsky, a soil microbiologist. In the
column, three main attributes of microorganisms are studied: oxygen, sulfur, and
photosynthetic ability. An oxygen gradient is formed by packing mud and essential sources of
carbon and sulfur in a tall container, kept in the dark for some time, and then allowed to remain
under a constant source of light and some heat. The column is continuously observed for
changes in color of the medium/suspension of mud, water, and essential nutrients. Both
aerobes (organisms using oxygen for respiration) and anaerobes (organisms that do not need
oxygen for respiration) are observed growing in colonies that have characteristic colors, such as
red, green, and purple. When you extract the unknown samples you will compare the
absorption spectrum to the known samples to determine if the colonies represent any of the
known photosynthetic organisms.
4 Lab Protocol: Making a Winogradsky Column
Materials:
• Rock-free mud
• Pond water
• Graduated cylinder or narrow, ridgeless water bottle
• Shredded newspaper
• Calcium sulfate/egg yolk
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Calcium carbonate/powdered chalk
Wooden dowel
Plastic wrap
Rubber band
Aluminum foil
*As you construct your Winogradsky column, consider one of the options below for your
investigation.
Option 1: Reproducibility
Test multiple bottles for each condition. For example, test three Winogradsky columns with egg
yolk. How reproducible are your results? Is there a lot of variation between the different
columns that were set up the same way?
Option 2: Independent Variable - Mud
Try testing several different sources of mud or soil to see if the microbial growth will be
different from location to location. You could even try some beach sand. What do you think
your results tell you about the soil quality and microbes that live at each site you test?
Option 3: Independent Variable - Additives
Test some different kinds of additives to look for microbes that live in unique and challenging
environments. For example, you could test increasing amounts of salt in a series of
Winogradsky columns to test for salt lovers (called halophiles) or place columns at different
temperatures to find microbes that like heat (near a heat vent) or cold (in the refrigerator). Can
you select for microbes that live in more extreme conditions?
Procedure:
1. Your teacher will provide rock-free mud as well as water from the same pond.
2. Obtain a 250 mL graduated cylinder and add about 1 sheet or 3 inches of shredded
newspaper (or paper towels) mixed with the pond water (or distilled water).
3. Mix about 250 grams of mud with 25 grams of calcium sulfate (or mashed hard-boiled
egg yolk) and 25 grams of calcium carbonate (or powdered chalk).
4. Add the above mixture to the shredded paper in the column till the cylinder is about
two-thirds full.
5. Using a wooden dowel or rod, tamp or pack the surface of the mixture tightly to
eliminate any air bubbles. Trapped air bubbles can alter the anaerobic environment in
the cylinder.
6. Add enough pond water to fill the cylinder to about 1 inch from the top and cover with a
piece of plastic wrap and a rubber band to keep it in place.
7. Wrap the cylinder entirely with aluminum foil to prevent any light from reaching the
contents. This will prevent the over-growth of algae and allow other organisms to grow.
8. Store the cylinder at room temperature for about 7 –10 days.
9. Remove the aluminum foil cover from the cylinder and place near a 60 watt bulb light
source for several weeks. Do not use fluorescent light sources.
5 10. Observe the column for growth. Different patches of color will appear. Also, check for
different odors. Note the color and position in the column on a weekly basis, and take
pictures with a camera as needed.
TITLE: The Winogradsky Column
SOURCE: Perry, et al., Microbial Life, First Edition, published by Sinauer Associates
© 2002 Sinauer Associates and Sumanas, Inc.
Explain:
Did the columns that were in the light make areas of green coloring on the sides facing the light
whereas the column in the dark remained dark brown?
Did the three columns that were in the light create color patterns that were somewhat different
from one another?
Over time gradients of different nutrients should have formed in the Winogradsky columns.
These gradients affect where different microbes grow within the columns. For example, over
time there's more oxygen at the top of a column that at the bottom, and this means that
microbes that can tolerate or make oxygen will be at the top. Microbes that cannot tolerate free
oxygen (called anaerobic bacteria) will be further down. Similarly, microbes that need light to
make energy (via photosynthesis or a similar process) will need to live where they can get light
in the column.
After about one to two weeks, depending on how much light the columns receive, some green
coloring should appear in the columns receiving light on the illuminated sides. This is mostly
due to cyanobacteria and algae, which need light. The column in the dark should remain dark
brown. In the column that had egg yolk you may have seen areas of darker green, purple,
and/or black coloring develop over time near the bottom—these colorings could be groups of
certain anaerobic bacteria: green sulfur bacteria, purple sulfur bacteria and sulfate-reducing
bacteria, respectively. Sulfate-reducing bacteria actually eat sulfur and make hydrogen sulfide
gas, which is eaten by the green and purple sulfur bacteria. In the column that had newspaper
you may have seen some areas of brown, orange, red or purple near the middle—these
colorings could be groups of purple nonsulfur bacteria, which need a carbon source to thrive.
You may have seen worms, snails, shrimp or other small organisms in the water, but probably
not many (if any) in the bottle with the egg yolk, because hydrogen sulfide is toxic to most
organisms!
6 Extend: Investigating Electrochemical Potential
Procedure:
1. Cut two lengths of bell cord wire, one long enough to reach the bottom of the column
and the other just long enough to touch the surface of the mud below the water layer.
2. Strip the last centimeter (cm) of insulation from the ends of each wire.
3. Place the long wire in the column so that the lower end rests at the bottom of the
column. Place the short wire so that it just reaches the mud water interface.
4. Bend both wires so that they hang over the top edge of the column.
5. Connect the wires to a multimeter set to measure voltage in millivolts (mV).
What is the source of the electrical current?
Is it chemical or photochemical? How could you decide?
Could the world energy crisis be cured by drawing energy from mud flats and bogs?
Explore:
How do you know what’s growing in your “pond”?
How might you confirm what species you have using spectroscopic techniques?
7 Lab Protocol: Chlorophyll extraction and absorption from known samples
Purpose:
To characterize the various types of chlorophyll used by organisms grown in a Winogradsky
column.
Background:
Colors form in the column due to bacteria-produced chlorophyll. Some of these bacteria can be
identified by analyzing the chlorophyll present in the column.
Materials:
• 1 developed Winogradsky column
• Disposable plastic pipette (10 or 25 mls)
• Eppendorf tubes
• Microfuge
• Vortexer
• Spectrophotometer
• Cuvettes
• Methanol
• Q-tips or small spatulas
• Squirt bottle with water
• Pasteur pipette
Procedure:
1. Study the Winogradsky column and identify an area to sample
a. Look for color differentiation
2. Cut the end off the disposable pipette to remove the tapered tip using a knife or
hacksaw
3. Insert the pipette into the Winogradsky column so as to capture different layers of
bacteria
4. While holding your finger over the end of the pipette, pull it out to remove a “core”
sample from the column
5. Clean the outside of the pipette with water and paper towels
6. Study the pipette to identify regions with distinct color
7. Using a gentle source of air on the top of the pipette, extrude the contents into a tray
8. Using a Q-tip or small spatula, transfer a sample the size of a pea of each of the
different colored samples each to an eppendorf tube
9. Resuspend the samples in 2 ml methanol and disperse using a glass stirring rod
10. Vortex for 30 seconds
a. Allow an incubation time of a few minutes for the pigments to extract into the
methanol
11. Centrifuge all tubes for 1 minute at high speed in the microfuge to pellet cell material
and debris
12. Transfer the supernatant liquid to a cuvette and measure the absorbance of the
different pigments on the spectrophotometer
13. Prepare samples from known bacterial cultures by centrifuging 1ml of cell suspension in
an eppendorf tube and discarding the spent media
14. Resuspend the cells in 2 ml methanol and process as above
15. Compare known samples with unknown samples
8 Explain:
A limiting factor for photosynthetic organisms is their light-harvesting efficiency, that is the
efficiency of their conversion of light energy to chemical energy. Small modifications or
variations of chlorophylls allow photosynthetic organisms to harvest sunlight at different
wavelengths. You used the absorption spectra of samples of microorganisms to identify the
organisms that developed in your pond ecosystem. These photosynthetic microorganisms are
the basis of much of the research of the Photosynthetic Antenna Research Center.
Oxygenic photosynthetic organisms usually utilize only the visible portion of the solar spectrum.
The cyanobacterium Acaryochloris marina carries out oxygenic photosynthesis but contains
mostly chlorophyll d and only traces of chlorophyll a. Chlorophyll d provides a potential selective
advantage because it enables Acaryochloris to use infrared light (700–750 nm) that is not
absorbed by chlorophyll a. Recently, an even more red-shifted chlorophyll termed chlorophyll f
has been reported. Scientists are now researching how to use modified chlorophylls to extend
the spectral region of light that drives photosynthetic organisms.
Evaluate:
What role do photosynthetic organisms play in an ecosystem?
How would changes to the population of photosynthetic organisms affect the population of
other organisms?
What microorganisms develop in a Winogradsky column and how does access to biotic and
abiotic factors influence their growth and development?
How are photosynthetic microorganisms different from plants that we are so familiar with?
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