Optimal Conditions for Proliferating Dinoflagellates Out of Their Natural Environment
I. Abstract:
To find the optimal conditions for maintaining and growing photo plankton that is a bioluminescent Dinoflagellate, "Pyrocystis Fusiformis", outside of their natural marine habitat. When they bio-illuminate, they emit a blue neon light. In lab, this light travels through a PETG (plastic) Erlenmeyer flask and essentially diffracts and slows down the velocity, wavelength, and frequency of the blue light, which my partner and I were set out to find also to determine if they grow more sufficiently in glass or plastic sterile bottles with our culture methods.
II. Introduction:
Pyrocystis Fusiformis emits a blue neon light when shaken in media out of their natural environment (the ocean) and when in a marine environment, they exhibit this feature for purposes of the 'Burglar-alarm theory'. When a predator nears and begins to attack, the masses of photo plankton illuminate to draw in the bigger prey. When the bigger prey nears, the bioluminescent algae's 'would-be-prey' know becomes the prey for the larger predator that the algae attracted.
In lab, p. fusiformis, produces bioluminescence on a circadian rhythm, being they need twelve hours of sunlight and twelve hours in the dark. While they are in the dark cycle, at about half way (6 hours in) they emit the blue neon light at their full potential. Remember, these algae are mixotrophs, meaning they can exhibit photosynthesis and heterotrophic metabolism. During the light cycle, if in sunlight, p. fusiformis, are the leading contributors of the atmospheres oxygen and acts as a major carbon sink that is very crucial for the worlds carbon cycle. Micro-sources (photon emitter) can be found throughout the cells cytoplasm that is primarily centered around the large vacuole. A single p. fusiformis cell can contain up to 4,500 micro-sources. Micro sources are composed of a round mass of vesicles which contain electon-dense short rods with rounded ends, sometimes crossed by electron-transparent narrow bands. Bioluminescence occurs when the protein luciferin is oxidized by the enzyme luciferase in the presence of ATP and oxygen, which all the reactions occur in the micro sources. P. fusiformis is used in mass amounts as a biological tool to determine the extent of contamination in a given area. They are easy to use for bioassays because they are inexpensive and simple in comparison to fish and other vertebrate species. They test the light emittance in a machine called 'QwikLite' after they have been in a contamination zone, and due to the light refraction in the QwikLite, we can determine the amount and type of toxin that the dinoflagellate was immersed in. They use them for toxicity readings on coastal regions containing powerplants, major consstruction zones, and military bases, due to the amount of chemicals that are washed out with rainfall into the ocean/estuaries.
III. Materials and Methods:
To start off, I had purchased a starter culture of p. fusiformis and algae grow which are the essential nutrients for algae that they need to sustain life and to continue growth. The starter culture was in a 150 mL erlenmyer flask, which was PETG poly-propyline. We then started the circadian rhythm with the starter culture, 12 hours in daylight, and 12 hours in the dark. Remember to keep them in between 65-75 degrees farenheit. After letting the starter culture grow and maintain a reliable circadian rhythm, after a week we then begin to make new cultures. We used a plastic sterile bottle and a glass sterile bottle for testing which material it will have optimal growth. Start with one plastic and one glass 500 mL sterile bottles. We only used 150 mL of salt water in each. To obtain the perfect salinity (gravity of 1.021-1.026). You can eaither measure out 30 grams of sea salt and add it to 1000 mL, after stirring the sea salt in the water you will want to pour a sample into the hydrometer to test the salinity and it should be within our preffered range for algae to grow. After the salinity is reached, test the pH to double check that it is within 7.2-7. 8 pH, although with the salinity being acheived the pH should correlate to it. For adding the algal culture and the algal grow, you can use any measurement of the algal culture you preffer, keep in mind the toal volume is only 150 mL so too much algaw from the culture may leave them with insufficient space to grow. We placed 10 mL of the algae culture into each the plastic and glass bottles that has 150 mL of salt water already in them. The ratio is 5:1 (5 parts algal grow to 1 part algal culture). After adding the portion of the algal culture and the algal grow to each the glass bottle and the plastic bottle, you will want to get an airator and connect the rubber tubes to it. Place a tube in each bottle that you have created and ensure that air is being pumped into each, but not too much air, only little bubbles are required, nothing too major. Once the new cultures are finished you will want to begin them on the circadian rhythm as well as continuing the starter culture in the rhythm as well. I was relying on indirect sunlight and using the hours of the day for their rhythm, but you are more than welcome to use a flourescent lamp on a timer or 12 hour incriments. I had left them growing for a month and examined them every night at about 6 hours into the dark cycle to ensure they are still growing and living in these conditions I had set up for them. When you tap, shake, move, or stir the contents in the bottles you will examine and brilliant blast of blue neon light that last for 5-10 seconds for one tap/shake. With continuous shaking you will be able to examine them until they dim out which will take a long while based on how much ATP and Luciferase is present in each cell. Eamine the light passing through the bottles and compare the brightness of the glass and plastic bottles and the PETG starter flask. The starter flask should still be the brightest due to having more cells. This is an amazing view and led me to calculating the frquency, wavelength, and velocity of the blue neon light as it is passing through the PETG vial. I am using the starter culture due to the brightness level and also the glass and plastic bottles were being used for 'what is a better container to grow algae in'. The main thing to remember is "the circadin cycle is crucial to maintain".
Blue neon light has a wavelength of 4.5x10^-7m (450nm) in a vacuum. With this and the help of light/wave equations used in physics and the usage of light deffraction equations and phase velocities you can calculate the speed of the neon light outside of a 'vacuum' in an environment and through a PETG erlenmyer flask.
IV. Results:
We all know the speed of light is 3.0*10^8 m/s and that it is constant and never changes or slows down, it only gets diffracted when it is passing through a certain media or surface. With knowing the speed of light and that the wavelength of neon blue light is 450nm (4.5*10^-7 m), the velocity (frquency) of the neon blue light can be found in a vacuum,
In a vacuum
frequency (blue light in vacuum) = (3.0*10^8m/s)/(4.5*10^-7m) = 6.5*10^14 Hz.
Energy = h*v = (6.63*10^-34Js)(6.5*10^14Hz) = 4.27*10^-19J
electron volts = There is 1.602*10^-19 Joules per 1 electron volt. e-V = (4.27*10^-19J)(1e-V/1.602*10^-19J) = 2.66 electron volts.
T (period in between each wave cycle) = (1/f) = (1)/(6.5*10^14Hz) = 1.5*10^-15 s
Phase velocity = (λ/T) = (4.5*10^-7 m)/(1.5*10^-15 s) = 3.0*10^8 m/s
Refractive Index = n = c/V(p) = (3.0*10^8 m/s)/(3.0*10^8 m/s) = 1