The interaction and sedimentation of the clay and bacterial cells within the marine realm, observed in natural environments, can be best investigated in a controlled lab environment. Here, we describe a detailed protocol, which outlines a novel method for measuring the sedimentation rate of clay and cyanobacterial floccules.
This method can help answer key questions in the field of sedimentology such as how organic rich, fine grain sediments can be deposited under oxygenated conditions. The main advantage of this technique is that both quantitative results and a visual record of sedimentation are produced. To begin, prepare Cyanobacterial cultures according to the text protocol.
Then rinse one 250 milliliter and two one-liter heat resistant Erlenmeyer flasks with four molar hydrochloric acid solution followed by distilled water. Fill the three flasks with liquid media. Seal the flasks with gas permeable foam stoppers and cover the necks with aluminum foil.
Then label and autoclave the flasks at 121 degrees Celsius and 100 kilopascals for 25 minutes. After this, allow them to cool to room temperature. To inoculate the media, flame sterilize an inoculation loop.
Then use the loop to transfer the Cyanobacterial culture into the 250 milliliter flask that contains 50 milliliters of medium. After inoculation, resterilize the lip of the 250 milliliter flask and replace the foam stopper and aluminum foil. Next, prepare the growth chamber as outlined in the text.
Transfer the inoculated 50 milliliter culture to the growth chamber and incubate with a maintained temperature of 30 degrees Celsius and agitation of 150 rpm. After successful culture growth, place the 50 milliliter culture and the one liter flask containing 400 milliliters of liquid medium in a laminar flow hood. Remove the foam stoppers and foil caps.
Use a Bunsen burner to sterilize the rim of each flask. Pour the culture from the 250 milliliter flask into the one liter flask containing 400 milliliters of medium. Then resterilize the neck of the one liter flask and attach the bubbling apparatus.
Finally, agitate the one liter flask at 30 degrees Celsius and 150 rpm with the bubbler apparatus attached to an air pump which circulates a humidified air mixture through the solution at the rate of 300 milliliters per minute. Incubate the culture for 120 to 168 hours at 30 degrees Celsius in the growth chamber. Using a graduated cylinder, transfer additional growth medium to the 400 milliliter culture until the final volume is one liter, thus diluting the culture.
Add the solution to an acrylic tank. Label two milliliter microcentrifuge tubes and place them near the acrylic tank. Then weigh out 50 grams of kaolin clay.
Use a camcorder to record the experiment. Pipette one milliliter of the Cyanobacterial sample into a labeled microcentrifuge tube. Use the sample to determine the Cyanobacterial cell count by measuring the optical density at 750 nanometers.
Quickly the pour clay into the tank while vigorously stirring with a stirring stick for 10 to 15 seconds. Then use a P1000 pipette to extract one milliliter of the sample for chlorophyll a determination and record this time as T0.Take additional samples at appropriate time intervals as outlined in the text. Next, use the modified procedure outlined in the text to determine the chlorophyll a concentration in each cell sample.
Use a microcentrifuge to pellet the cells from each sample at room temperature for three minutes at 13, 000 times gravity. Then use a P1000 pipette to remove the excess medium. Resuspend the cell pellet with one milliliter of 100%methanol.
Incubate the samples at negative 20 degrees Celsius for 24 hours. After centrifuging the samples again, use a spectrophotometer to determine the chlorophyll a concentration. This experiment illustrates the effect of clay on Cyanobacterial populations and their settling rates.
Cultures of the Cyanobacteria Synechococcus that are mixed with kaolin clay are seen to have higher settling rates than the cultures containing only Cyanobacteria. After being exposed to the clay, Cyanobacterial cells settle out of suspension within 10 minutes. Cultures of the Cyanobacteria Synechocystis mixed with clay are also seen to have higher settling rates than cultures containing only Cyanobacteria.
After being exposed to clay, these Cyanobacterial cells are also brought out of suspension in 10 minutes. After watching this video, you should have a good understanding of how to measure sedimentation rates of Cyanobacteria and clay mixtures which can be used to inform depositional models of black shells.
