The Students: Jessica Byrd, April Haskins, and Betsy Shotton
The Teacher: Clinton A. Kennedy
After the big Biocoil was constructed by the Sewage Sisters a few years ago, people expressed interest in growing their own algae in a home version of the Biocoil to harvest and eat. For the past two years we have been attempting to create a marketable Biocoil system that will produce a pure-strain algae. This could be of interest to people who would rather grow their own algae than buy algae that has been harvested from a lake, dried, and bottled. We are currently growing chlorella because that is the algae being used in the big Biocoil.
Algae are single-celled organisms and simple multicellular plants that live in colonies wherever there is water-- the sea, fresh water sources (such as lakes and ponds), moist soil, and in the bodies of animals. Algae are differentiated mainly by cell structure, composition of pigment, nature of the food reserve, and the presence, quantity, and structure of flagella. The following phyla, or divisions, are recognized: blue-green algae; euglenids; yellow-green and golden-brown algae; dinoflagellates and similar types; red algae; green algae; and brown algae. However, we'll be paying specific attention to the green algae Chlorella, a member of the "green foods" (chlorophyll-containing foods that are rich sources of essential nutrients).
Chlorella is the algae we are growing in our photosynthetic bioreactor. It is a two-and-a-half billion year old, single-celled algae found in fresh water that reproduces at one of the fastest rates of any living plant. It is the first known form of plant life with a true nucleus. Chlorella has a higher amount of chlorophyll per given volume than any other known plant. In addition to chlorophyll, chlorella contains vitamins, minerals, dietary fiber, nucleic acids, amino acids, enzymes, and Chlorella Growth Factor (a group of substances that has the potential to help repair damaged organs and tissues).
Chlorella has enough carbohydrates to make it a virtually complete nutrient. The biochemical make-up of the chlorella cell consists of nutrients that closely match the nutrient needs of a human cell, which may explain its cell-protective properties. Of all green foods, chlorella possesses the highest amount of nucleic acids, which contain concentrations of RNA and DNA. Chlorella is a complete protein food that contains all the B vitamins (one tablespoon of dried chlorella powder provides 333% of the RDA vitamin B-12), vitamin C and E, and many minerals high enough to be considered supplementary amounts. The cell wall of chlorella has a positive effect on intestinal and bowel health, detoxifying the colon, stimulating peristaltic activity, and promoting the growth of beneficial bacteria while having some antiseptic action against the growth of harmful bacteria.
Chlorella is known as the "King of Alkaline Forming Foods," as it helps to maintain a good pH balance. Chlorella is effective in its ability to detoxify the body of heavy metals including lead, mercury, copper, and cadmium because it strengthens the liver, the body's main detoxifying organ. Chlorella may offer a degree of protection against toxic pollutants including radiation, due to the fact that it has six times more beta carotene than spinach, which has been said to effectively combat UV radiation. In addition to beta carotene, chlorella contains more iron than spinach. Chlorella has been known to reduce arthritic stiffness, lower blood pressure, and relieve gastritis and ulcers. Chlorella has been effective in weight loss programs because of its rich nutritional contents-- both in cleansing ability and in maintaining muscle tone during lower food intake. A further benefit is a noticeable increase in energy and health when consuming chlorella regularly. Of the over 25,000 species of algae, chlorella offers the most nutritional benefits because it contains 1.7 - 7.0% natural chlorophyll, which can help cleanse the body.
Scientists consider chlorella to be one of the most promising agents in the treatment of cancer. In a series of studies, scientists showed that cancerous growths in mice could be reduced and even stopped by injecting a water solution of chlorella around the neoplastic growth. In some cases, tumor cells were killed outright at the point of chlorella injection. Later, it was discovered that chlorella, given in the oral form, had the same anti tumor effect.
While it is popular in most areas of the world, chlorella has been slow to catch on in the United States. From the back of a "broken cell chlorella" bottle of 100 (410 mg) capsules, we read that consumers should swallow three capsules daily with water--one at each meal. Therefore, eating this particular kind of chlorella introduces approximately 1230 mg into the diet. A bottle of chlorella at GNC costs $16.95, and we figure that, at three capsules a day for one person, people would be spending this amount for chlorella every month. Thus, if we can design our photosynthetic bioreactor to produce 1230 mg (or more) per day and still be cost friendly, it would be a product of interest to algae-lovers.
Sea water contains minute traces of inorganic and organic nutrients that are essential for the growth of phytoplankton (such as algae), on which all life in the oceans depends. The concentration of these nutrients is generally less than 1 part per million of sea water.
The most important inorganic nutrients for algae growth are nitrogen and phosphorus, generally present as nitrate or ammonia and as phosphate, respectively.
Nitrate is an organic ion that occurs naturally as part of the nitrogen cycle. The nitrogen cycle contains four processes: ammonification, nitrification, denitrification, and fixation. Denitrification converts fixed nitrogen back into the unusable, gaseous nitrogen state. The other three convert gaseous nitrogen into usable forms. The process we're concerned with is fixation.
Nitrogen fixation is the conversion of nitrogen in its gaseous state to ammonia or nitrate. A series of different microorganisms accomplish the fixation process. Some free-living aerobic bacteria freely fix nitrogen in the soil. Blue-green algae can fix nitrogen in both the soil and water, yielding ammonia as the stable end product.
Nitrate concentrations higher than 45 mg/liter may cause methemoglobinemia (Blue Baby Syndrome). In methemoglobinemia, the reduction of nitrate to nitrite results in toxicity of nitrates in humans. Nitrite forms methemoglobin, a substance that does not bind and transport oxygen to tissues, when it reacts with hemoglobin. The formation of methemoglobin may lead to asphyxia (suffocation due to lack of oxygen). Since methemoglobin accounts for 1 - 2% of the globin in the body, a methemoglobin level greater than 3% is defined as methemoglobinemia. In other words, when nitrate becomes nitrite in the body, it reacts with globin to form methemoglobin, which does not transport oxygen to the cells. Without oxygen, suffocation occurs.
Symptoms of early or chronic toxicity (early or chronic nitrate poisoning) in animals include: watery eyes, reduced appetite, dirty appearance, weight loss or no weight gain, and signs of vitamin A deficiency. Signs of acute toxicity include: accelerated pulse rate, labored breathing or shortness of breath, muscle tremors, weakness, staggering gait, cyanosis (some membranes, such as the tongue and the whites of the eyes, turn blue), and eventually death.
If a nitrogen limited system is supplied with high levels of nitrogen, significant increases in algae production may occur. This can be a positive or negative effect. Because we are trying to grow algae, we are aiming for high levels of nitrate: however, high levels of nitrate could be harmful to consumers of the algae grown in our photosynthetic bioreactor if the Chlorella does not effectively remove the nitrate. Nitrogen levels generally control the rate of primary production. Thus, if the system is supplied with large levels of nitrogen, algal blooms will occur. The recommended level of nitrogen in estuaries to avoid algal blooms is 0.1 to 1.0 mg/liter, while the phosphorous concentration is .01 to .10 mg/liter.
Phosphorous is an essential nutrient for all life forms. It plays a role in DNA, RNA, ADP, and ATP. Phosphorous is required in order for these necessary components of life to occur. In fresh water marine systems, phosphorous exists in either a particulate phase or dissolved phase. Particulate matter includes living and dead plankton. Dissolved phosphorous is digested by phytoplankton and altered to organic phosphorus. The phytoplankton are then ingested by zooplankton, which excrete it as inorganic phosphorous, and the cycle continues.
Phosphate itself does not have notable adverse health effects. However, phosphate levels greater than 1.0 mg/liter have been known to interfere with coagulation in water treatment plants. As a result, particles that harbor microorganisms may not be completely removed before distribution.
Phosphorous is generally the limiting nutrient in fresh water aquatic systems. In other words, if all phosphorous is used, plant growth will cease despite the amount of nitrogen still available. On the other hand, if sufficient phosphorous is available, nitrate concentrations will rise, and algal blooms will occur.
A phosphorus deficiency is rare, except in people with certain gastrointestinal malabsorption syndromes, such as Crohn's Disease. A mild deficiency causes fatigue, weakness, and a decreased attention span. A severe deficiency may lead to seizures, coma, or even death.
Water contains both hydrogen (H+) ions and hydroxyl (OH-) ions. The pH test measures the hydrogen ion concentration of substances and gives them a pH value on a scale that ranges from 0 to 14. If a water sample has more hydrogen than hydroxyl ions, it is acidic and has a pH less than 7; the more acidic the sample is, the lower the number is. If the sample contains more hydroxyl ions than hydrogen ions, it is basic with a pH greater than 7; the higher the number is, the more basic the sample is. A neutral sample, such as pure deionized water, contains equal numbers of hydrogen ions and hydroxyl ions and has a pH of 7, neither acidic nor basic. For every one unit change on the pH scale, there is approximately a ten-fold change on the pH scale; that is, a pH of 4 is ten times more acidic than a pH of 5 and one hundred times more acidic than a pH of 6. This is represented with the formula: pH = -log[H30+]. A good range of pH for algae is typically between 5.5 and 10.
Invented by Lee Robinson, the founder of the British biotechnological company Biotechna, the Biocoil is a "photosynthetic bioreactor that provides an environment for biological organism to grow in a controlled manner." [Sewage Sisters] Our photosynthetic bioreactor, however, will serve a different purpose than the one the Sewage Sisters developed, but in order to build our model, it is necessary for us to understand how theirs worked.
The frame is built and clear PVC tubing is wrapped around it to form a circular model so that it is easier for photosynthesis to occur. Sunlight or artificial light is then put in at an angle to shine on the tubing while the algae flows through. Chlorella algae was used in the Sewage Sister Biocoil to remove nutrients from the sewage flowing along with the algae.
The tubing of the Biocoil consists of several sections of tubing rather than one piece wrapped all of the way around. Compressed air pumps are used to push the algae and water through the Biocoil and to prevent anoxic conditions in the water. To clean the Biocoil system, the direction of the pump is reversed, and a scouring pad "pig" is sent through to remove algae build-up from the sides of the tubing. The system is cleaned regularly because the algae build-up could prevent light from getting to the algae, and therefore preventing photosynthesis from occurring.
In order for the algae to grow and use nutrients, photosynthesis has to occur. Photosynthesis is the biological process by which the energy of sunlight is absorbed and used to power the formation of organic compounds from carbon dioxide and water. Although photosynthesis is usually associated with green plants, it does occur in algae. Ultimately, photosynthesis supplies all living organisms with the energy needed to survive. Photosynthesis can be summed up in one basic formula:
sunlight + carbon dioxide + water ---> organic compounds + oxygen
Thus, we must account for the fact that we need carbon dioxide, water, and light to run our photosynthetic bioreactor.
After demolishing last year's photosynthetic bioreactor and salvaging any reusable parts, we set to work redesigning a new and improved prototype. We began by asking ourselves "Where in their house would a person want to put a photosynthetic bioreactor?" We agreed that a photosynthetic bioreactor would most likely end up in a garage or basement and that it needed to take up as little space as possible. After much discussion, we agreed that last year's model was too "bulky", and a tall, thin model would be more practical than a short, squat model because it would take up less space.
We calculated that it would take about four feet of photosynthetic bioreactor at a twelve inch diameter to equal the amount of tubing used in last year's photosynthetic bioreactor. We had to design a "base" to support our photosynthetic bioreactor and decided that an enclosed box would be the most attractive and efficient way to not only keep the apparatus organized but also sterile. In a tentative design, we made the box 20" x 18."
We wanted a tank to put inside this box that funneled at the bottom to avoid any algae settling at the bottom (a problem from last year), but couldn't find one that would hold more than one gallon of water. A quick look at our box led us to believe that with connectors and the addition of a shelf, we could make our design work with two one-gallon tanks rather than with just one two-gallon tank.
Our next step was to carefully calculate and design the entire set up of the inside of our box to ensure that we had the room to include all the necessary parts for the photosynthetic bioreactor (Co2 pump, gravity fed pump, two tanks, four posts, connectors, feed tube, and a harvesting outlet). We decided that our original 20" x 18" box would work, but because of the thickness of the plywood, it would actually be 18" x 20 3/4". We designed a shelf, complete with holes for the tanks and posts to support the photosynthetic bioreactor, down 10 1/2" from the top of
With our design complete, it was time to start building. We measured and traced the various pieces onto the wood that we would need to cut for our box. We recruited Stan Aschenbrenner to cut our eight-foot 2' x 4's lengthwise (so that we had four eight-foot 2' x 2's) and to cut out the pieces of our box. To cut the holes in our shelf for our tanks, we took a spare jug and cut off the top. We traced the outline of the jug in two places on the piece that was to be the shelf of the box where we wanted the jugs to fit, and Stanley cut out the holes. The holes for the posts were a bigger problem, though; we thought that if we traced the ends of the posts onto the wood and cut the holes out, the posts would fit snugly into our shelf. However, when we tried to fit the posts in the holes, we couldn't slide the shelf far enough down and ended up cracking it in the process. With a new piece of board for the shelf, we again traced the holes for the tanks and cut them out. For the posts, however, we cut out squares, deciding that the posts did not have to fit perfectly inside of their appropriate holes. We screwed the pieces of the box together.
In order for the water in the photosynthetic bioreactor to be able to drain into the tanks, we had to drill connectors into the tanks--one at the top and one into the lid. Mr. Funkhouser was kind enough to drill these holes for us using the drill press. As one of the holes was slightly too small for the connector to fit into it, Mr. Funkhouser beveled the connectors to fit. We used aquarium sealant to secure the connectors and ensure that water would not leak out.
For our feeding tube, we attached a Dr. Pepper bottle to a piece of one-inch (inside diameter) tube. We found that a lid from a peanut butter jar perfectly capped-off the open end of the Dr. Pepper bottle, preventing any excess air from entering our system and contaminating the Chlorella.
We mounted our lights by bracing them with two pieces of wood screwed to the uprights on the inside of the coils. We added handles to the sides of the box for easy lifting. We attached the spigot and sealed the two smaller tubes for the CO2 pump onto the jugs and sealed the feed tube to the left jug. We hooked up our tubing to the spigot and put the end through the top hole. We wrapped and snap-tied our coils around the posts.
We added our algae water and ran into a problem when we realized we needed to pig it out (send a small piece of foam through the tubes to scrape the algae off the sides), but hadn't taken that into account when we designed it. We could put the pig in down by the pump, but in order to take it out, we had to cut our drain tube back into the tanks and hook it together with a connector and a pig stop (a pin through the tube that prevents the pig from going into the tank).
We have started running tests. Our most recent pH was one of 10, so we have to do some more research to find a safe way to lower our pH. We've tried to do agar plates to test for bacteria, but we ended up with bacteria in the bottom of our control dish as well as our variable dishes, leading us to believe that our "sterile" petri dishes weren't sterile. We used a nichrome wire to scrape the bacteria out and looked at the sample under a microscope. The slides looked like they contained cocci bacteria. A recent algae density test yielded 11 grams of algae per liter of water.
Plans and Goals
We are working on developing a testing schedule to test for our variables: pH, Nitrate, Phosphate, algae density, amount of algae water we need to harvest to yield at least one gram of algae a day, how much nitrate and phosphate we need to feed our algae, and how fast our algae uses these nutrients. We plan on pigging our photosynthetic bioreactor out every Monday or Tuesday we have class so that it gets pigged at least once a week. We will test for pH everyday we have class on Thursday (every other week).
Our first testing procedure will be for algae density. For a week we'll take out 200 mL of algae water to dry in the autoclave. For now, we're not worried about dissolved solids. During the week, we'll check the algae density to see if it's the same after we take out 200 mL. We'll need to replace the water we take out. We'll dry and weigh the algae everyday to make sure that the amount of algae we dry out doesn't decline. If 200 mL doesn't yield a gram a day, we'll experiment with larger amounts. If we're not pulling out the same amount of algae everyday, we'll have to experiment with different amounts of nutrients.
We need to know how much algae we need to produce and how fast we need to produce it. Based on recommended consumption of chlorella capsules in a bottle of chlorella at GNC, we need to produce at least 1230 mg of algae per day per person. Of course, depending on what people want to put the chlorella in (shakes, algae water, etc.) we may need to produce more. We need to be able to produce this amount of algae everyday while maintaining a constant algae density. We need to experiment and determine how much of the nutrients we have to feed the Biocoil so the algae can keep up. We also need to make sure that the algae uses up all these nutrients. Greg Moller from the University of Idaho has volunteered to test our samples for nitrate/nitrite. We must set safety guidelines for our variables. We need to test for bacteria and plan on asking a hospital to help us run a blood agar test. We need to find out what happens when we overfeed our photosynthetic bioreactor in case people who have photosynthetic bioreactors in their homes feed it too much. We need to experiment with different filter systems to successfully separate our total dissolved solids and our total suspended solids. We plan to check with our city water provider to find out about potential contaminants in the tap water people may want to put in the photosynthetic bioreactor.
Because our main goal is to one day market our photosynthetic bioreactor, we must take into consideration what people want. We have composed a survey and plan to put it in health food stores in the hopes that we can get some feedback from consumers.
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