Lab #5 Multicellular Marine Primary Producers. Part 1: Photosynthesis and Photosynthetic Pigments

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1 Lab #5 Multicellular Marine Primary Producers Part 1: Photosynthesis and Photosynthetic Pigments Introduction Photosynthesis is a fundamental life process upon which all living things depend. Organisms that practice photosynthesis are using energy from the sun to produce food (glucose). The basic equation for photosynthesis is as follows: 6CO 2 + 6H 2 O C 6 H 12 O 6 + 6O 2. This process is accomplished by a large number of enzymes and pigments which are usually contained within an organelle, the chloroplast. In the simplest explanation, the chloroplast captures energy from the sun and uses it to combine carbon dioxide and water to form sugars (food) and oxygen. Of course, it is an incredible complex process involving many steps and many enzymes and pigments. The pigments used in photosynthesis can be categorized into three groups, chlorophylls, carotenoids and phycobilins. Chlorophyll a is central to the absorption of light energy and can be found in all photosynthesizing plants and algae. Since chlorophyll a can absorb only a small number of the available wavelengths from the sun, other pigments serve as accessories by collecting additional wavelengths and transferring the energy to chlorophyll a. Other pigments aid photosynthesis by protecting the essential pigments from excessive sun damage. Different taxonomic groups (red algae, brown algae, green algae.) utilize different accessory pigments and those pigments can vary in quantity within the taxonomic groups resulting in the huge variety of color amongst photosynthesizing organisms. The energy emitted from the sun comes in a full spectrum of wavelengths. The visible portion of this spectrum can be further divided into specific colors which correspond to specific wavelengths. For example the wavelengths between 500 and 550 nanometers appear green in color. Additionally, the visible wavelengths of light are used by plants for photosynthesis. In the ocean, photosynthesis is a challenge because some of the visible wavelengths of light are quickly absorbed by the sea water. Only the blue wavelengths of light penetrate deeper than the first few meters and coincidentally are not very useful for photosynthesis. This explains the abundance of photosynthesizing organisms in the shallow subtidal and their steep decline in abundance as you move deeper into the ocean. Seaweeds have developed specific adaptations to deal with this challenge including the production of pigments to harvest the limited light available as well as morphological adaptations. Kelps utilize air bladders to float to the surface where more light is available and kelps and other seaweeds produce large blades to maximize light absorption. We will investigate the variety of pigments found within photosynthesizing organisms. Initially we must extract the pigments from the seaweed and then we can utilize chromatography to visualize them. Extraction The method we will use to extract the pigments from seaweeds is the hyperosmotic shock method. The photosynthetic pigments of the seaweeds are contained and embedded within the chloroplast membranes as well as the plasma membrane. These membranes need to be broken down to release the pigments and this can be done mechanically by grinding the seaweeds with brute force or hyperosmotically by exposing the cells to a solution that is so salty

2 that the water rushes out of the cells and the membranes collapse. We will use this latter procedure because it is more convenient. Follow the instructions below. 1. Obtain a 20g sample of your specimen (red algae, brown algae, green algae or plant). 2. Cut your specimen into pieces no larger than 1cm 2 and place them in a 100ml beaker. 3. Add enough 6M NaCl solution to your beaker to completely submerge your sample. 4. Wait 5 minutes. 5. Remove your specimen from the beaker and blot it dry. 6. Clean and dry your beaker. 7. Replace your specimen in the clean & dry beaker and pour enough 95% methanol into the beaker to just cover your sample be very careful not to add too much methanol! 8. Place it immediately into the dark for 30 minutes. 9. Pour the extract into a vial with a lid or a small beaker covered with aluminum foil and place on ice to preserve the pigments for further use. Throw the seaweed tissue into the trash and clean your glassware. Chromatography Chromatography is a technique in which molecules in a solution can be separated and visualized. Fortunately, pigment molecules already have color, so they are easy to see once they are separated. The technique we will employ to separate them relies on their differences in molecular weight and polarity. We will place a sample of each extract on a strip of paper and place it vertically in a large test tube with a small amount of solvent at the bottom. As the solvent is absorbed into and up the paper, it will dissolve and carry the pigments up with it. The smaller pigments will be carried up faster than the large pigments and when the solvent has reached the top of the paper, the pigments will have separated out. Follow the steps below to separate the pigments of your specimen. 1. Obtain a strip of chromatography paper, a capillary tube, a large test tube and a beaker to set the test tube in. 2. Place the tip of your capillary tube into your extract and then place that tip onto a spot 1cm from the tip of your chromatography paper, see image below. 3. Wave your paper in the air until the spot dries. 4. Repeat application of extract and drying 5 more times. 5. Place 1 ml of solvent in the bottom of your test tube. 6. Place the chromatography strip in the test tube point down and replace the stopper. 7. Place the test tube in the beaker and allow it to sit unmoved until you can see that the solvent has moved to within 5 milimeters of the top edge. 8. Remove the strip and place it on your counter until it has dried (approx. 1 minute) 9. Identify the pigment bands using the chart below and record the distance traveled for each pigment on your data sheet. Pigment Name Chlorophyll a Chlorophyll b Chlorophyll c Carotene Xanthophylls Fucoxanthin Color on strip Blue-green Yellow-green Light green Orange Brown Orange-brown

3 Sketch the bands on the chromatography paper below Chromatograph for the red algae Chromatograph for the brown algae Chromatograph for the green algae Chromatograph for the plant Label the pigment bands on the chromatograph above and enter the distance they traveled (mm) in the chart below. Use the information provided on the previous page to identify your bands. Pigment Name Red alga Brown alga Green alga Plant Chlorophyll a Chlorophyll b Chlorophyll c Carotene Xanthophylls Fucoxanthin Which pigment is the smallest?

4 Part 2: Survey of Multicellular Marine Primary Producers Introduction The photosynthesizing organisms of the ocean come in a variety of forms and sizes. They are members of the Kingdom (Domain) Bacteria, Kingdom Plantae, and Kingdom Protista. They include microscopic organisms like cyanobacteria and dinoflagellates as well as large rockweeds and enormous kelps. The classification of the multicellular algae is controversial, and your textbook has included them in the Kingdom Protista that is an antiquated group. There are fundamental differences among the groups of algae that suggest they belong to more than one kingdom. Our understanding of the relationships among the Protista and other kingdoms is growing rapidly with the use of molecular data, but no final organization has yet been agreed upon. Rather than burden you with the current ambiguity, we will use the classification used in your textbook with the understanding that it will soon change. The multicellular algae are found in shallow aquatic habitats worldwide and commonly referred to as seaweeds. These seaweeds can be subdivided into the red algae (Division Rhodophyta), green algae (Division Chlorophyta) and brown algae (Division Phaeophyta). The red and brown algae are found almost exclusively in marine environments. These organisms as well as the green algae create a significant habitat for many invertebrate animals (crabs, worms, shrimps, etc.) as well as fish and some mammals. Additionally, they contribute to their local food chains both as a direct food source to herbivores and, as they dissolve, food for bacteria and small zooplankton. Each group of seaweeds has its own very unique set of photosynthetic pigments that result in their distinctive colors. In addition each seaweed group has unique cell wall components, energy storage molecules and chloroplast structure indicating they do not share a recent common ancestor. There are relatively few species of the Kingdom Plantae in the ocean, and they are represented by the seagrasses here on our coast. You may have a chance to look at a few specimens of seagrass in class. These organisms differ from the algae not only in that they have vascular tissue to transport water and nutrients but they also produce fruits and flowers. The members of the Kingdom Plantae all use a very similar set of photosynthetic pigments resulting in their distinctive green color and they share this suite of pigments in common with the green algae suggesting a close evolutionary relationship. Procedure You will examine representative specimens of brown, green, and red algae along with seagrasses (if possible). These may be live, preserved, or herbarium specimens. If the specimen is alive, place it in a glass dish on a sheet of white paper. Be sure to keep the specimen moist. Preserved specimens must remain in their sealed containers. If you are working with herbarium specimens, study them on a flat surface. Avoid bending the preparation. Keep them horizontal when moving them from one location to another. Sketch the specimens and note their sizes, shapes, colors, and conspicuous structures. It is very important that you make your own observations and your own drawings. It is unacceptable to copy someone else s drawing.

5 Scientific name: Scientific name: Phylum / Division: Phylum / Division: Scientific name: Scientific name: Phylum / Division: Phylum / Division:

6 Scientific name: Scientific name: Phylum / Division: Phylum / Division: Scientific name: Scientific name: Phylum / Division: Phylum / Division:

7 Questions 1. What is the function of the: holdfast? blade? air bladder? 2. Describe two ways seaweeds participate in food webs. 3. How are seagrasses different from green algae? 4. How are seagrasses similar to green algae? 5. Please use a short description or simple sketch to define the following terms used to describe seaweed morphologies. Blade - Filament - Branching - Coralline - Crustose -

8 Part 3: Products Derived From Algae As you learned in lecture, many products that we use in our daily lives have ingredients that are derived from algae. Take a look at the products that have been brought into the lab for you and note the division from which they come. Products that are derived from brown algae (Phaeophyta) Products that are derived from red algae (Rhodophyta) Are there any products derived from green algae? What word(s) should you look for on an ingredient list to determine if the product contains a brown-algal derivative? What word(s) should you look for on an ingredient list to determine if the product contains a redalgal derivative?

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