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http://www.greenstarusa.com/news/08-01-31A.html, 10-8-09: 11.52

GSPI Has Acquired A License For Next Generation Algae To Biofuels Process Efficient Production of Both Biodiesel And EthanolCan Now Be Achieved Through Algae SAN DIEGO(BUSINESS WIRE)(E-WIRE) January 31, 2008 Today, Green Star Products, Inc. (OTC: GSPI), announced that it has acquired a license to utilize a breakthrough processing technology to convert algae biomass to feedstock oil and cellulose sugars for the production of biodiesel and cellulosic ethanol respectively.

The new process uses an efficient low-cost method to extract the oil and cellulose sugars from oil-bearing microalgae that eliminates the need to mechanically dry and press-extract the algae oil using traditional methods. The sugars from carbohydrate-rich cellulose and hemicellulose can be used to make a variety of products including ethanol and other high demand chemical products. The oil can be made into biodiesel and other products.

The removal of oil from the microscopic algae has been a stumbling block for the commercial production of fuel from algae for many years.

GSPI has secured the technology license from Biotech Research, Inc. (BTR), one of GSPIs consortium partners. The process continuously strips the oil from the algae and also reduces its biomass into different carbon chain carbohydrates, proteins and other constituents. BTRs intellectual property is protected by patent pending status.

Joseph LaStella, president of Green Star, stated, "GSPI along with a handful of other high tech companies are leading the industry in algae commercialization; however, there are two major hurdles to overcome: First, an efficient, affordable construction and processing method to control the environment to promote optimum algae growth; Second, efficient harvesting and extraction of oil from the microscopic algae biomass".

Green Star, along with Biotech Research, has been operating one of the largest demonstration algae facilities since April 2007. Phase I and II testing were successfully completed in 2007. The results of Phase III have been completed and announced today (January 31, 2008) under a separate Green Star press release titled GSPI Completes Algae to Biodiesel Winter Demo Testing.

The Montana facility has clearly demonstrated a solution to the first problem, i.e. an affordable method to grow algae, and now GSPI has potentially solved the second hurdle the low-cost extraction and conversion of microalgae biomass to oil and other useful products.

Biotech Research, Inc. operates a high tech research facility at the University of Baja California in Ensenada, Mexico (see four-minute video at GreenStarUSA.com), where a team of scientists and engineers are studying short, medium and long-term technologies for the advancement of algae production.

Mr. LaStella further stated, To limit algae research to the production of fuels is a grossly short-sighted view point. Algae have the answer to many of the global problems facing us today. Our old microalgae friends have been around for three billion years and were responsible for creating the oxygen atmosphere we now breathe. Algae grow as much as 100 times faster than agricultural crops, so algae could potentially solve all of our food and environmental problems.

Algae can reverse our Global Warming problems; provide unlimited biodiesel and cellulosic ethanol; provide high protein food for the World's increasing population; be used as feedstock for an unlimited number of industry products and chemicals; and, the list goes on.

Biotech Research, Inc. is researching a host of algae uses. Some of these uses can be a bit surprising. For instance, Solazyme, Inc., recently announced an algae joint venture with Chevron (NYSE: CVX) and a breakthrough using an algae strain that can reproduce itself without sunlight.

Biotech Research is also involved in "algae that grows in the dark"; however, it is not BTRs top priority research project for the following reasons:

1. Algae that grow without sunlight do not use the photosynthesis process; therefore, dark-growing algae need expensive food sources like sugars, vitamins, etc. to survive and reproduce. This means that it is not obtaining energy from sunlight and it is actually not consuming CO2 but producing CO2 like any other animal or burning process. CO2 mitigation is not possible with these algae strains. The need to increase photosynthetic processes for CO2 sequestration is a major reason why there is interest in algae farm development (see press release titled Green Star States: U.S. Industry Gets Serious About Cutting CO2 Emissions from December 20, 2007).

2. Since grow in the dark algae do not use the (free) energy from the sun they must get their energy from something else, mainly sugars that are poured directly into the growing algae medium. Where is all this sugar going to come from? Back to agricultural crops?

Biotech Research has the real answer: Making oil and sugars from photosynthesis grown microalgae biomass and non-food biomass that can be derived from a variety of agricultural and municipal waste streams (wood chips, corn cobs, switch-grass, etc.).

Mr. LaStella further stated, It should be understood that the success of this new process is not required for the first generation of algae production. First generation algae production can produce 4,000 gallons of oil per acre per year (versus 50 to 100 gallons for other oil crops) and later generations will produce 10,000 gallons or more per acre.

Green Star Products and Biotech Research are also researching independently, and in coordination with other technology companies, additional high tech processing systems to convert biomass algae into usable fuels and products. These products and systems include:

1. Direct pyrolysis

2. Advanced mechanical extraction

3. Separation of sugar from biomass carbohydrate chains

4. Hybrid fuels

5. Low temperature fuels

6. Enzyme extraction

7. Algae strain development

8. High efficient LED artificial light production

9. Natural algae growing enhancers

And many other proprietary technologies.

Today, algae and non-food biomass technologies are the most likely tools to change our world on a grand scale.

Mr. LaStella further commented that, "The U.S. Congress recently (Dec. 19, 2007) passed a huge energy bill into law, which contains billions of dollars to support the production of ethanol from non-food sources (cellulosic ethanol). GSPIs new licensed process combines feedstock algae production into biodiesel and cellulosic ethanol, which qualifies our new process for financial support under the new energy bill."

Please also read GSPIs other press release issued today (January 31, 2008) titled GSPI Completes Algae to Biodiesel Winter Demo Testing.

Green Star Products, Inc. (OTC:GSPI) (OTC:GSPI.PK) is an environmentally friendly company dedicated to creating innovative cost-effective products to improve the quality of life and clean up the environment. Green Star Products and its Consortium are involved in the production of green sustainable goods including renewable resources like algae biodiesel and clean-burning biofuels, cellulosic ethanol and other products, as well as lubricants, additives and devices that reduce emissions and improve fuel economy in vehicles, machinery and power plants. For more information, see Green Star Products' Web site at http://www.GreenStarUSA.com, or call Investor Relations at 619-864-4010, or fax 619-789-4743, or email [email protected]. Information about trading prices and volume can be obtained at several Internet sites, including http://www.pinksheets.com, http://www.bloomberg.com and http://www.bigcharts.com under the ticker symbol "GSPI".

Forward-looking statements in the release are made pursuant to the "safe harbor" provisions of the Private Securities Litigation Reform Act of 1995. Investors are cautioned that such forward-looking statements involve risks and uncertainties, including without limitation, continued acceptance of the company's products, increased levels of competition for the company, new products and technological changes, the company's dependence on third-party suppliers, and other risks detailed from time to time in the company's periodic filings with the Securities and Exchange Commission. CONTACT:Green Star Products, Inc.Joseph LaStella, President619-864-4010 619-789-4743 [email protected]

Last Updated: January 31, 2008

http://www.hydrodrive.co.in/HYDRODRIVE%20ALGAE%20FUEL%20TECHNOLOGY.htm, 10-8-09: 12.04

HYDRODRIVE ALGAE GREEN FUEL TECHNOLOGY:

HYDRODRIVE ALGAE GREEN FUEL TECHNOLOGY makes use of a technology patented in Great Britain, India and rights protected in the USA, CANADA, JAPAN, CHINA, PHILIPPINES and in several countries involving " A PROCESS AND SYNTHESIZER FOR MOLECULAR ENGINEERING OF MATERIALS (Great Britain Patent No. GB 2397782,India Patent No.200286) to produce HIGH CETANE GREEN SYNTHETIC DIESEL from ALGAE at the most economical cost.WHAT MAKES ALGAE -THE FUTURE FOR HIGH CETANEGREEN SYNTHETIC DIESEL FUEL?.Algae can be found almost everywhere oceans, ponds, swimming pools, and common goldfish bowls. While algae are not truly plants, these single-celled organisms have the same photosynthetic ability to convert sunlight into chemical energy. The various species are Blue Green Algae, Filamentous Algae, Pond Algae, Horsehair Algae, Toxic Algae, Algae Diatoms, Green Algae, Brown Algae, Pond Moss, Pond Scum. For some species of algae, this chemical energy is in the form of oils very similar to common vegetable oil.Over the last 20 years microalgae production volumes have increased strongly. The cultivation of microalgaeis is proven to be the most profitable business in the biotechnology industry. It is a wasteless, ecologically pure, energy- and resource-saving process. Microalgae are a diverse group of microscopic plants with a wide range of physiological and biochemical characteristics and contain, among other things, high quantities of natural proteins, enzymes, amino acids, pigments, 30% lipids, over 40% glycerol, up to 8-10% carotene and a fairly high concentration of vitamins B1, B2, B3, B6, B12, E, K, D etc, compared with other plants or animals. Moreover, microalgae are important raw materials for amino acids, and other medically important products.Microalgae, like higher plants, produce and store lipids in the form of triacyglycerols (TAGs). TAGs could be used to produce a wide variety of chemicals, i.e.fatty acid methyl esters (FAMEs), which can be used as a substitute for fossil fuel-derived diesel. This fuel, known as biodiesel, can be synthesised from TAGs via a simple transesterification reaction in the presence of acid or base and methanol. Algae have emerged as one of the most promising sources especially for biodiesel production for the main reasons: The yields of oil from algae are orders of magnitude higher than those for normal oilseeds. Algae can be grown away from farms and forests, thus minimising the damage caused to the eco and food chain systems. They are also harvested very quickly, dramatically speeding up production process.The oils from algae is processed and used to produce HIGH CETANE SYNTHETIC GREEN DIESEL with the patented HYDRODRIVE'S SYNTHESIZER in the EUROPE and the QUALITY OF FUEL stands tested to be far superior than the petrodiesel. .ALGAE DO NOT GRAB FOOD CULTIVATION LAND AND INFLATE FOOD, EDIBLE OIL PRICES:Algaes single-celled structure is extremely efficient in using sun light and absorption of nutrients. Algaes growth and productivity is 30 to 100 times higher than crops like soybeans, rapeseed or jetropha.Algae production does not compete with agriculture. Algae production facilities are in closed enclosures and do not require soil for growth. Algae use 99% less water than conventional agriculture. Algae can be located on non-agricultural land far from water. Whole organism in algae converts sunlight into oil. Algae can produce more oil in an area the size of a two-car garage than an entire football field of soybeans/corn/rapeseed or jetropha.Algae can be grown in sewage and next to power-plant/cement plant smokestacks where they digest the pollutants to produce oil.To produce the required amount of biodiesel by growing soybeans would require almost 3bn acres of soybeans fields, or over 1bn acres of canola fields at nominal yields of 48 and 127 gallons of oil per acre, respectively. Conversely to produce 15,000 gallons of oil per acre from algae would require only approximately 9.5m acres. Microalgae grow much faster than the land grown plants, often 100 times faster;ALGAE OIL PRODUCTION IS INFINITELY SCALEABLE:The right naturally occurring algae species can, under just the right conditions, produce oil at near-theoretical limits. Their small size less than 30 microns and aquatic nature makes them ideal for a large-scale, highly automated, closed production system called a PHOTO BIO REACTOR. Microalgae have uniform cell structures with no bark, stems, branches or leaves, allowing easier extraction of products and higher utilisation of microalgae cells. Large scale systems are highly-tuned to provide each cell the precise conditions needed for maximum productivity with light and carbon di oxide sensors for faster multiplication and yield. The cellular uniformity of microalgae makes it practical to manipulate and control growing conditions for the optimization of cell properties. This means that even land not suitable for farming can be used to grow algae. Furthermore, this may be beneficial to countries not capable of raising crops due to their economy; the relative cheapness of growing biodiesel algae could be a saviour for them. ALGAE EAT AND DIGEST GLOBAL WARMING CARBON DI OXIDE, NITROGEN DIOXIDE, EXHAUST GASES AND POLLUTANTS:Algae live on a high concentration of carbon dioxide-the GREEN HOUSE GAS (GHG), nitrogen dioxide (NO2)-a pollutant of power plants and diesel exhaust. These pollutants in the atmosphere from the automobiles, cement plants, breweries, fertilizer plants, steel plants are nutrients for the algae. Algae production facilities can thus be fed with the exhaust gases from fossil fuels of these plants to significantly increase productivity and clean up the air.It is known that the biological method is considered the most eff ective and economically efficient manner for the purification of industrial wastewater by using the microbiological active slime and algae. However, bacteria of the active slime have low stability to high concentrations of organic and mineral components. This method also requires further destruction of superfluous quantities of active slime, which also contains other pathogenic microorganisms. Microalgae on the other hand possess higher stability, which enables their use in more concentrated and toxic environments. One specific species of algae utilises mineral elements, spirits, sugar, and amino acids, and compared to active slime, enables higher purification rates up to 96-98% for organic and 80% for mineral components. It also has organic acids which prevent the growth of pathogenic microorganisms in solution. For example, a chemical plant in europe demonstrated high levels of cleaning of its phenol wastewaters from this specific algae species. Similar observations have been made at a nitric acid fertilizer and sugar plants, as well as cattle-breeding and poultry farming establishment. Sewage derived raw materials, which at present pollute the environment, and simultaneously provides biological clearing for these wastewaters creating an additional source of profit is possible with algae.ALGAE OIL BYPRODUCTS:The Oxygen can be used for the hospitals and industries. The carbohydrates remaining after the oil has been extracted from the algae and can be used to make animal feed. During 2007, the primary goal was to increase the feed assimilability, but it was achievable principally by using small concentrations of powdered activated carbon and adding enzymes, raising only the degree of cellulose hydrolysis, assimilability and the commodity weight of production per feed unit. This one-sided approach has resulted in product quality impairment and a decrease in animal resistance to illnesses. Furthermore, an acute increase of frequency of mass epidemics among animals and poultry in various countries was observed. This has caused great economic damage to manufacturers and whole countries. The manufacture of vaccines against mass epidemics requires enormous feats of organization and is not always effective. A notable example was a new strain of H5N1 avian flu virus, which, at the end of 2006, was detected in China and was resistant to previously-produced vaccines.Another problem faced today is the consequences caused by the over-use of antibiotics in animal feed. While antibiotics were proven to be effective in improving poultry production, their use came under pressure as an increasing number of consumers feared that their inclusion in animal feed rations would lead to antibiotic resistant bacteria that are pathogenic to humans.In 2005 the EU removed the last antibiotic growth promoters from pig and poultry diets. The search for alternatives to these additives continues to attract intense interest. As consensus begins to develop among the scientific community on this subject, a few approaches stand out in terms of efficacy, technological and economical feasibility, particularly in terms of organic acids and the use of essential or botanical oils. Organic acids provide a natural alternative, reducing production of toxic components by bacteria and causing a change in the morphology of the intestinal wall that reduces colonization of pathogens, thus preventing damage to the epithelial cells. Anions of organic acids deactivate the RNA transferase enzyme, which damage the nucleic acid multiplication process and eventually result in death of the organisms. But the use of organic acids and essential oils in the feed industry are potentially a source of other problems: corrosion, worker safety, handling, vitamin stability in pre-mixes, environmental concerns, and the stability of products.

With all this in mind, the use of algae as a feed additive could become the best solution, since microalgae contain natural organic acids that reduce colonization of pathogens. Thanks to this feature, of a specific species of algae towards feed conservation and reduction of microbiological pollution of wastewaters.Some specific species of algae possesses other biologically attractive priorities, such as: A high concentration of chlorophyll (5-10 times) Chlorophyll is an effective means for the treatment of anaemia, pancreatitis, skin ulcers and diabetes. A unique cell wall which consists of three layers; a middle part consists of cellulose, and the outer layer is formed of polymeric carotene which is capable of adsorbing toxic elements and removing them from organisms. High contents of vitamins, especially pro-vitamin A carotene which not only plays an important role during the growth process, but destroys cancer cells in their initial stages and improves the generation of macrobacteriophage in the immune system.An ability to intensively synthesize high concentration of nucleonic acids with a combination of high contents of fibres, peptides, amino acids, vitamins, sugars and trace elements. Not only does this promote rapid reproduction of algae, but as a growth factor also provides favourable conditions for algae use in other organisms.The potential poultry demand for microalgae powder (as feed additives) is US$8.8m in the Armenian domestic market, more than US$1.2-7.2b in the US, more than US$1.4bn in China, and US$600m in Iran.The specific algae is microscopic, green, single cell organism with a diameter of 3-10m. During 12 hours the cell undergoes four-fold reproduction in optimum conditions. Compared to traditional plants, water consumption is 10-times lower. The biomass yield per unit area is five times higher.ALGAE FOR CEMENT AND POWER PLANTS POLLUTION CONTROL:In general, traditional large scale biomass sources are not yet practical for the cement and power plant industry. Furthermore, not all biomass sources are available all the year-round for this application. The exhaust steam and effluent gases emitted from cement and thermoelectric power plants could be used for microalgae suspension heating in pools and biomass all year round. During microalgae aeration of effluent gases, CO2 is turned into O2 by photosynthesis, further potentially reducing industrial CO2 industrial emissionsMicroalgae production and its biomass use for biofuel industry has global prospects and may provide sustainable economic development. It is possible to expect that in the near future algae will solve fuel problems and also will improve the quality of life of farmers, thus leading to a global re-orientation of priorities for fuel production.Microalgae production may turn out to be a truly global way to settle global warming problems and farmers poverty problems in all developing countries.WHAT HYDRODRIVE SYNTHESIZER DO WITH ALGAE BIOFUEL?:HYDRODRIVE SYNTHESIZER makes use of the patented process to synthesis the algae biofuel upon excitation by waves resulting in plasma catalysis yielding HIGH CETANE GREEN SYNTHETIC DIESEL with better cloud point, changed physical properties such as HIGH CETANE INDEX above 80 with excellent combustion and emission properties such as NOx free emission than the petro diesel at a cost very much less than the conventional petro diesel and also for use as an additive to improve the existing petro diesel qualities.http://www.advancedaquarist.com/issues/aug2002/breeder.htm, 10-8-09: 13.44THE BREEDER'S NET by FRANK MARINI, Ph. D.Sponsored in part by:

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This is the first in a series of microculture columns in which well explore the culture of larval fish foods. In this months column, we focus on phytoplankton, the basic nutritional building block of our home fish breeding effort. Well survey useful types of phytoplankton, discuss why they are important in our home fish breeding efforts, and examine what it takes for home culture of these microalgae

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We are what we eat When it comes to raising larval fish, nutrition is one of the most critical factors to success. For fish fry, the principal step in the food chain is phytoplankton, and while it is rare that we feed phytoplankton directly to larval fish, we utilize phytoplankton to enrich a food items -- rotifers, copepods, brine shrimp nauplii, etc. These enriched food items are fed directly to larvae. Rotifers, Artemia nauplii, and copepods that are depleted of essential nutrients, have little, if any food valve, and therefore it is critical that we dont ignore the role of providing these nutrients via phytoplankton. Due to the fact that food items take on a similar nutritional value as the phytoplankton cells that they consume, the nutritional value of the phytoplankton is of paramount importance to success with our larval fish.

Phytoplankton are simple, unicellular organisms capable of photosynthesis. Unlike higher plants that are composed of multiple cells and differentiated tissues, phytoplankton lack a stem, any type of roots, or leaves. Because photosynthetic organisms manufacture their own food, they form the basic energy source that sustains many natural food chains. These plants are the starting point.

So what makes phytoplankton so nutritious? The focal point of nutrients in these microalgaes is the concentrations of omega-3 fatty unsaturated fatty acids (HUFAs). Numerous studies have shown that marine fish are unable to synthesize sufficient quantities of two essential HUFAs; Eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) [Kanazawa, 1979.] These two fatty acids are essential in the growth and development of fish. In general terms, the higher the level of HUFAs, the more nutritious the phytoplankton are to fish.

The Microalgae Appropriate microalgae that are readily cultivable at home, and are suitable food for prey items which will be fed to fish fry are critical to our home breeding success. There are approximately 7000 species of microalgae, although many of which are not adaptable for home culture. Currently a few species are readily available and readily adaptable to home culture: Nannochloropsis oculata, Chaetoceros gracilis, Isochyrsis galbana, and Tetraselmis sp. The nutritional value of each of these microalgae vary, which makes some species more appropriate for our use than others. {Table 1}. In the following paragraphs I will describe several useful phytoplankton species.

Table 1 Comparison of phytoplankton discussed in the text

AlgaeTotal HUFAEPADHA

N. Oculata16-43%HighLow

C. Gracilis5-11.5%0.3-2.5%

I. Galbana2-4%3-4.2%

T. Iso0.2-0.7%8.3-11%

Tetraselmis~5%~6%

Nannochloropsis oculata is a 2-4 micron (m) green flagellate. This is a fast growing species that is easy to maintain. This phytoplankton is the one most commonly thought of when the term green-water is used. This is a dark green alga with a thick tough cell wall that interestingly is readily consumed by rotifers. N. Oculata is high in overall omega-3 HUFAs (ranging from 16-42%), and while most of the HUFAs are composed of EPA, there is little DHA present. A growth study performed by Okauchi et al [Okauchi 1990] determined that the highest level of EPA was attained at 7 days after batch cultures were inoculated. N. oculata has been shown to contain very high levels of vitamin B12, which is critical for larval fish survival, and it has also been suggested that vitamin B12 is important for developing diseases resistance in larval fish as well.

Chaetocerous gracilis is a 6-9 m solitary diatom with four large spines. It is frequently used in large quantities in commercial shrimp culturing. Because of its protruding spines it has been suggested that this phytoplankton can be problematic in rearing food items; however, this problem has never borne out in commercial cultures. C gracilis has an EPA range from 5-11% EPA and DHA from 0.4-2.5%.

Isochrysis galbana is a 4-7 m golden-brown flagellate. This species is commonly used in bivalve culture (clams, oysters, etc). While it has been occasionally used as a single rotifer food, it is usually mixed with other phytoplankton such as chlorella or N. ocultus. The EPA levels range from 2-3.5% and DHA is 3.5-4%. Different strains of this species have varying levels of HUFAs, and one isolate found off Tahiti (commonly known as T-Iso) contains high DHA (8-11%) and low EPA (0.2-0.7%). This EPA level is much lower than found in a standard reference strain of I.galbana. An important note for home culture is that this strain requires consistent temperatures, vitamin additives to the nutrient broths and a silicate additive to reach maximum density. According to Wilkerson [Wilkerson, 1998] this algae is too temperamental, fragile, and fastidious to be used regularly.

Commercially available phytoplankton. A perfectly acceptable alternative to home culture of phytoplankton is to purchase commercially grown phytoplanktons. Here are two 1 gallon containers of velvet green (N.oculata) + rotifers (www.mountaincorals.com). These commercially available phytoplankton preparations offer the ease and convienence of high densities phytoplanktons in a minimal culture media. Unfortunately these products have a limited shelf life, and need to be kept cool to maintain nutritional content. Shown here is a clownfish fry grow out tank setup of James Wiseman.

Close up of the phytoplankton bottles. This culture was recently harvested so you can see the inside. Notice the rigid airline tubing extending to the bottom of the container and the stream of large bubbles, ofimportance is that there is no frothing or skimming on the culture surface. Photo courtesy of Joe Burger.

Tetraselmis sp. is a 9-14 m motile green flagellate, which has been successfully used in outdoor ponds because it is extremely temperature tolerant. There are several species of Tetraselmis sp. that are available and one such T. tetrathele is frequently used in aquaculture. Studies have shown that while EPA (~ 5%) and DHA (~7%) levels in this phyto are theoretically sufficient, several authors has suggested that rotifers feed diets exclusively on T. tetrahele were not capable of sustaining fish larvae [Fukusho 1985, Wilkerson 1998]. To combat this deficit, aqua-culturists have fed mixtures of T. tetrahele with other phytoplankton species and discovered that these combinations were significantly more nutritious than those cultured alone. Of interest to hobbyists, Tetraselmis sp, produces two antibiotic-like compounds which have been documented to increase survival in larval fish feed on prey items enriched with this phytoplankton.

Given the above information on the available phytoplankton, which one is the best to use? Considering primarily ease of growth and sufficient HUFA profiles, N. oculata is my first choice, followed by C.gracilis and I. galbana, and lastly T-iso. While C. gracilsis and I. galbana has similar nutritional profiles, C. gracilis grows more rapidly and more consistently in culture. Another important consideration is which of these phytoplankton will grow under home water conditions. Each phytoplankton species and strain has an optimum pH and salinity range in which it grows best. It is only through experimentation that you will discover which one grows best for you. A chart of optimal parameters is provided to allow you to make some comparison {Table 2} [Wilkerson 1998 pg157].

Table 2: Optimal conditions for phytoplankton discussed in the text

AlgaeOptimal pHTemp RangeMinimum Illumination (LUX)Salinity

N. Oculata7.0-8.460-864,000-5,00022-25

I. Galbana7.8-8.577-861,000-6,00028

Tetraselmis6.968-821,000-20,00030-40

According to commercial experts, rarely is the use of a single phytoplankton suitable for aquaculture of fish larvae. Nutritional deficiencies found in one phytoplankton species can be compensated for by adding another phytoplankton species superior in that missing HUFA. As an example; N. oculata which is high in EPA, but low in DHA can be paired with T-Iso, which is high in DHA. Some hobbyists even add a small portion of Tetraselmis to this co-culture just to add an antibiotic effect. Studies performed in commercial fisheries have shown that fish larvae fed prey items enriched on diets composed of multiple phytoplankton species have higher survival rates and quicker growth rates than those larvae fed food items enriched with a single type of phytoplankton. The take home message here is that the use of multiple phytoplankton species is advantageous. If you must only use a single culture of phytoplankton use the one with the highest HUFA concentrations.

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A commercially available culture reactor produced by AB Aqualine. This 2L vessel is designed to accept air or Co2 into the lower port, and the central drain allows removal of the green-water .

So what are the basic phytoplankton growth requirements? Phytoplankton are much like more familiar plants, and have three simple needs: nutrients, water, and light. Of course each element has tremendous impact on the growth and nutritional profile of your phytoplankton. Optimizing each aspect will increase your success. Plants require nitrogen and phosphorous, along with some trace elements (such as zinc, iron, etc) and vitamins (B12, thiamin, etc). For water, the home aquaculturist must provide clean, buffered, artificial saltwater. For home phytoplankton culture, pay specific attention to the appropriate salinity range (specific gravity 1.014-1.017), and the appropriate pH (7-8.5). Finally, provide adequate lighting by supplying a light source which gives an intensity of 1000-10,000 Lux on the cultures. Of course, this light can be generated from a variety of light sources, from simple fluorescent tubes to intense metal halide bulbs.

Now lets get into specifics One of the best sources to obtain all your home phytoplankton culture products is Florida Aqua Farms, Inc. (see the shopping list). This company not only has all the phytoplankton starter cultures, but also provides all the fertilizer mixtures, culture containers, and one of the best resources on growing phytoplanktons. This book is entitled The Plankton Culture Manual by Frank Hoff and Thomas Snell. If you want to read and learn basic through advanced phytoplankton culturing techniques, this is the book for you.

The phytoplankton: Phytoplankton cultures can be purchased from online suppliers. These cultures contain either live phytoplanktons growing in a nutrient solution, then shipped suspended in a liquid, or as a live phytoplankton cultured in a semi-solid agar medium. These are called algae wafers (see the shopping list) and will last for 2-3 months in a cool environment. Essentially all the phytoplankton species we discussed above are available in wafer form.

Culture containers: The simplest culture container is a clean 2 or 3 liter soda bottle made of clear plastic. Round-bottomed bottles (as opposed to the more common dimpled-bottomed ones) work best, as they allow better water circulation. One important note about the use of any container for phytoplankton culture regards sanitization or sterilization. Sterility is a demanding standard, and means that the culture vessel and medium are absolutely devoid of all forms of life. For our purposes, we can adopt a lower standard: no previous exposure to any phytoplankton, phytoplankton predators or competitors. A sufficiently sanitized soda bottle is one that has been emptied of the soda, rinsed with uncontaminated water, and left to dry upside down. You can store excess bottles dried and capped for future use. After you start culturing phytoplanktons in these bottles, you will notice a greenish film buildup on the inside. This film buildup will prevent light penetration and would need to be removed before reusing the bottle. While cleaning this dirty bottle with a dilute muriatic acid rinse will remove the green film, I find it easier to just buy another 2L soda bottle and not bother with the acid wash.

Aeration: The water in the phytoplankton culture must be adequately aerated. Aeration allows proper mixing of air and carbon dioxide in the culture. Aeration will also help stabilize the pH of the culture and maintain a uniform distribution of phytoplankton cells. Airstones are not required in microalgal cultures. In fact, fine bubbles can be detrimental to your culture as phytoplankton can be trapped at air-water interfaces. A rigid, open-ended airline tube is superior to a diffuser for this application. A simple air bubbler can be created by obtaining a three-foot long 1/8 rigid air tube. This air tube can be cut in lengths that reach to the bottom of your culture vessel (the soda bottle), at the top of this rigid airline you attach the soft flexible airline, which comes from your pump or gang valve. An interesting point about aeration in soda bottle is that you can often determine the quality of your culture by how it bubbles: an old or damaged culture will often produce foam (like that seen from a protein skimmer). You can also detect if youre over-aerating or have a nutrient depleted (crashed) culture as this will also result in foaming.

Nutrients: As I had mentioned above phytoplankton require nutrients and the simplest way to provide these nutrients is through the use of fertilizers. The fertilizer Guillard formulation is common f/2 (f/2 = 1/2 full strength, full strength is listed as f) http://www.florida-aqua-farms.com/Section04/FAFw4.htm and is easily available. This mixture is a concentrated stock solution of essential elements and trace elements. In our home phytoplankton cultures we will be adding 1-3 milliliters of f/2 per liter of culture.

Lighting: One of the easiest and inexpensive lighting sources is a Home Depot 48 dual bulb shop light. This seven-dollar light fixture will support 2-48 long 40 watt fluorescent bulbs. An adequate bulb for home phytoplankton culture is the GE-F40 DX. This is a 6500K bulb, which costs three dollars and emits more than sufficient light for our needs. These 48 long shop lights will allow us to use 7 2-liter soda bottles in a row as our culture station, while a 24 bulb will easily house 4 2-liter bottles.

Required Equipment for a basic phytoplankton culture

Algae: a liquid culture (such as DTs) or an algae disk, (i.e., Nannochloropsis algae disk)

Fertilizer: 1 bottle of Microalgae Grow (f/2 nutrients)

Culture containers: 2-8 cleaned, rinsed, with caps, 2liter soda bottle (round bottom perferred)

Lighting: inexpensive Home Depot shop light (dual 48 bulbs), or a dual bulb 24 inch

2-4 fluorescent bulbs (6500K is sufficient)

Aeration: 1 or 2 high-powered air pumps, either with 2 or 4 outlets, controllable. The more bottles you have the more air youll need.

2 or 4 outlet gang valve(s), metal or brass tends to work better than cheap plastic ones. Sufficient 1/8 flexible airline tubing, 1-2 3 foot long rigid airline tubing.

Other items: Aquarium salt, pH strips, salinity hydrometer, filter floss, measuring cups, plastic dropper pipettes, plastic medical syringe (1-10mls: for measuring volumes of fertilizer).

Where to begin: There are a number of online websites that offer a do-it-yourself (DIY) version of green-water culture stations (see DIY sites below). These sites provide a good visual representation of what will be described below. Additionally, there are a number of written resources which describe green-water culture in detail [Hoff, 1987 Moe 1989, Toonen 1996].

The first time making a new culture medium, it is important to use new saltwater and a fresh culture container. New saltwater means just that: never-been-used-for-anything water and a clean, dried, and capped 2L soda bottle. This is not used tank water or saltwater you bought at a local fish store and stored away. Our concern is that bacteria will be present in this water and these containments will readily overtake your phytoplankton cultures. A simple method that can be used to pasteurize water is to boil it in a microwave. However, I use fresh out of the tap water mixed with a salt mix and dechlorinated. A recipe recommended by Wilkerson is 3/8-cup aquarium salt, 1-gallon tap water, 3 milliters (mL) of MicroAlgae grow (f/2). [Authors note: I actually prefer to use 1/2 mL of f/2 per liter (or 1mL/2L bottle)]. According to Wilkerson a good reason to use tap water is that tap water contains nitrates, phosphates and metals that are beneficial in phytoplankton culturing. However, I have also used water obtained from a reverse osmosis unit, and this has also worked well. To begin the actual culture, use your starter culture (the algae wafer or a few milliliters of your liquid suspension) and add it to your fresh culture medium. One algae wafer can be used to start a two 2L bottle of culture medium. After adding the algae to the culture medium, you will notice a light green tint. This is what you want. Place the culture bottles within 2-3 inches of your light source and add the air bubbler. Adjust the air bubbles to between 10-20 air bubbles/sec. After the airline is placed, add a wad of filter floss to the bottle opening to prevent any contamination. Within five to ten days you will observe a rich green algae growth (or brown is you use T-iso.) From this culture you will start new bottles. A word of caution here is to be patient; often the starting cultures will require a few extra days to reach its dark green potential. Remove a few milliliters of green-water from the existing culture to seed the new bottles. Again, you want a slight green tint to the new culture. As you continually remove culture medium from the bottles, add new fresh culture media to the existing culture. Once you have established a sufficient number of green-water bottles (at least 3 to 5) you can start harvesting your green-water to feed your prey items (rotifers, brine shrimp nauplii, copepods).

4 bottle phytoplankton culture station. This arrangement utilizes 2-24bulbs, and 4 2 liter soda bottles. Note the culture containers are marked with the date of inoculation, and labeled. The upper shelf houses the phytoplankton and the bottle shelf contains the rotifer culture. Photo courtesy of Joe Burger www.cnidarianreef.comClose up of 2L soda bottle phytoplankton culture containers. Using this arrangement, the soda bottle caps were drilled and rigid airline tubing was inserted. To ensure optimal aeration make sure the rigid tubing goes to the bottom of the soda bottle. If all the vessels contain approximately the same amount of liquid you will get equal distribution of air bubbles. Shown here are 4 new starter cultures of phytoplankton. Photo courtesy of Scubadude http://www.coralfragz.com

Next lets discuss some tips that will allow us to maximize our phytoplankton yields and accelerate their growth. The first parameter to focus on is lighting. If we provide lighting for 16 hours daily, we should get the best growth. Remember cellular growth and protein production occurs during the plants dark cycle and if we were to illuminate for 24hrs we would not increase our yield of phytoplanktons proportionally. It is particularly important to know that many species of phytoplanktons poorly tolerate 24 hour photoperiods. While the cultures dont crash, they fail to grow to expected levels. Some commercial firms utilize 24hr illumination, but they also have light barriers in their cultures. The phytoplankton cultures are exposed to the light side 1/2 the day and then pass behind the light barrier into darkness for half the time. So these revolving cultures have a built-in dark cycle. In our case, 16 hours of light and eight hours of dark is sufficient for N. oculata. Next, to ensure that we obtain optimal concentrations of green-water, we should count the number of phytoplankton cells per milliliter of medium. This procedure is simple to perform, but it requires a microscope, or a calibrated cell counter. For visual cell counting, simply remove one ml of culture media, and then take a drop from this. Place the drop on a slide and count, you dont have to count every single cells, but the idea here is to get an rough estimate between having a few cells per drop and a lot of cells per drop. To the human eye, a dark green phytoplankton culture may only have 10-20 thousand cells/mL, but to others dark green might correspond to 100-200 thousand cells per mL. Quick cell counting is a ball park method to get an idea of how many cells/mL are present, and to train your eyes to better estimate your phytoplankton concentration. While youre counting cells, make sure to scan for contamination. Not surprisingly, cyanobacterial contamination will often make your green-water cultures appear much more lush. Next we monitor pH and maintain a useful pH throughout the culture process. A fully grown green-water culture will have a high pH (a culture containing >100 thousand cells/mL can be easily around pH 9-10). Advanced hobbyists may chose to utilize CO2 bubbling in their cultures to maximize the growth of green-water and maintain a proper pH. However for the average hobbyist, since we will be maintaining cultures containing 20-100 thousand cells/mL, the pH will remain around 8.4-8.6. I would recommend monitoring pH and salinity in your cultures to ensure youve obtained the appropriate values. The reason for maintaining proper pH and salinity is that these green-water cultures will be used directly in feeding prey items (like rotifers) and having vast differences in the pH and salinity between the rotifer stocks and your phytoplankton media often results in shocked food items.

To maximize our green-water production and to ensure we can feed our food items (rotifers, etc) lets start to rotate cultures. Mark the culture bottles with letters (eg. A, B, C and D) and start your cultures. Once all the bottles become dark green, harvest 2/3 of A, then add 2/3 new culture media to this bottle, the next day remove 2/3 of B, and refill w/ new culture media, then on the next day remove 2/3 of D and repeat. Using this pattern you will feed two-thirds of a bottle to your food items daily and restart the green-water cultures from the remaining 1/3. Once you have completed this cycle culture A should be ready for harvest. The key is to leave behind a sufficient amount of rapidly growing green-water which will inoculate this new culture. Since you only have to regrow 2/3s of the new bottle, this occurs quite rapidly.

I would be remiss if I didnt include a few key tips that will boost the nutritional levels of the phytoplanktons. There are a number of factors that will influence the levels of HUFAs in our home production of phytoplanktons, and we can pay particular attention to these as we optimize our production. Phytoplanktons are most nutritious when their growth is still within the exponential growth phase. This is before the culture is saturated, and growth rates begin to roll off. Ultimately, the culture will enter what is called stationary phase and the algae will be much less nutritious. If our standard technique is followed, the exponential growth is maintained for approximately 7 days from when the culture was first inoculated. Phytoplankton cultures past this exponential phase move into the stationary phase (days 8-10) of growth and begin to decrease their nutritional values. A second tip is that the growth medium has a significant impact on the quality of the phytoplankton. While the standard phytoplankton fertilizer f/2, a report by Wilfors [Wilfors, 1992] determined that changing any of the components of the f/2 resulted in a significant change in the biochemical composition of the phytoplanktons and had the resulting effects of decreased survival of fry which fed on food item enriched by this. F/2 is readily available from a few sources Ive identified in the shopping list. One word of warning, do not omit ingredients from or otherwise change the f/2 composition.

Of course there are alternatives to growing your own phytoplankton. A great advancement for hobbyists is the availability of aquacultured phytoplanktons. N. oculata is available from some sources as live cultures. This product is commonly known as DTs phytoplankton and is a concentrated stock of enriched phytoplankton in a minimal culture media. When stored in cool conditions, this green-water product has a nominal 2 to 3 month shelf life. While purchasing liter-to-gallon containers of this green-water allows you to bypass any home culture or expansion, what you save in time you pay for in dollars. However, for a busy hobbyist this is a perfectly acceptable alternative. Other phytoplankton species are also becoming available to the hobbyist as live mass cultures.

There are also a number of green-water alternatives that can be used in place of live phytoplankton to enrich prey items. These alternative foods attempt to provide similar levels of nutrients to the prey items, and take advantage of rotifers ability to consume a number of similar size particles. Hopefully this months column has given you a fresh interest in culturing your own green-water and has ignited your interest in at home breeding of fish. In next months column well discuss rotifer culture and the use of both phytoplanktons and alternative foods for enrichment. We will also discuss hatching Artemia nauplii and ciliate culture.

Shown here is the phytoplankton culture after the left most bottle has been harvested. As you can see in the photo there is a large color difference between the freshly started culture and that of the rapidly growing cultures. (bottles 2, 4, and 5 L-R ). Photo courtesy of Joe burger.

Ill conclude by suggesting you go thru the link list provided below and check out some of the online suppliers of these aquaculture products. This will leave you with plenty to do until next month, when we again peer through The Breeders Net.

Online suppliers

AQUACULTURE SUPPLY, LLC. 668 Time Saver Avenue New Orleans, LA 70123-3144 U.S.A.Telephone: 504-736-9360 FAX: 504-736-9373Aquatic supply house http://www.aquaculture-supply.com/Aquacenter 166 Seven oaks Rd Leland MS 38756 800-748-8921 http://www.tecinfo.com/~aqcenter/

(source for f/2, culture containers, enrichments products) Aquatic Eco-systems Inc http://www.aquaticeco.com/ (source for f/2, culture vessels) Brine Shrimp Direct P.O. Box 13147 Ogden, UT 84412-3147 U.S.A. 800-303-7914 toll-free http://www.brineshrimpdirect.com/ Carolina Biological Supply, 2700york rd Burlington, NC 27215 800-334-5551 http://www.carolina.com

(source for phytoplankton starter cultures, culture vessels, nutrient mediums)DTs Marine PhytoplanktonsHamshire Il 60140

Phone 847-683-4564

(source for commercial N. Oculata)Florida Aqua-Farms Inc33418 Old Saint Joe Road Dade City, FL 33525 Phone: 352-567-0226

http://www.florida-aqua-farms.com/

(source for f/2, phytoplankton cultures, measuring dip sticks, reading material)Northeast brine shrimp: http://216.25.82.220/nebs.htmProvosoli-Guillard National Center for Culture of Marine Phytoplankton(CCMP)http://ccmp.bigelow.org/

(source of living stock cultures collection of marine phytoplankton)SACHS Systems Aquaculture : http://www.aquaculturestore.com/University of Texas-Algae culture site: http://www.bio.utexas.edu/research/utex/

(excellent scientific source for information/background on algae, also sells phytoplankton starter cultures)DIY green-water culture stations : http://reefkeeping.com/issues/2002-07/ds/index.htmhttp://community.webshots.com/user/matthewbeaman Matt Beaman, an advanced hobbyist who has tremendous practical experiencing growing phytoplanktons and food items

http://www.sjwilson.net/reef/ Flame Angels Homepage: a hobbyist who has a nice DIY green-water reactor and food item grow-out tanks

Additional reading

Hoff, F.H., Snell, T.W., Plankton Culture Manual, Florida Aqua Farms Inc; ISBN: 0966296001; 5th Rev edition (July 1999)

Wilkerson, J.D., Clownfishes, Microcosm Limited; ISBN: 1890087041; (June 1998) Rearing a Plankton Menagerie by Shawn Carlson http://www.sas.org/E-Bulletin/2001-10-19/labNotes/labNotes.html

References: Cripes, D., Algae Nutrition, J MaquaCulture 7(3): 57-64. 1999.

Fukusho K, Okauchi M, Tanaka H, Kraisingdecha P, Wahyuni S, Watanbe T., Food Value of the Small S-strain of Rotifer Brachionus picatilis cultured with Tetraselmis tetrahele for the Larvae of Black Sea Bream. Bull Natl Res Inst Aquacult 8:5-13, 1985.

Hoff, F.H., Snell, T.W. Plankton Culture Manual, Florida Aqua Farms, Inc.; ISBN: 0966296001; 5th Rev edition, 1999.

Kanazawa A, Teshima S, Kazuo O., Relationship Between Essential Fatty Acid Requirements of Aquatic animals and the Capacity for Bioconversion of Linolenic Acid to Highly Unsaturated Fatty Acids. Comp. Biochem Physiol 63:295-298. 1979

Moe, M., The Marine Aquarium Reference: System and Invertebrates, Green Turtle Publications, Plantation, Florida; 1989.

Okauchi M, zhou W, zou W, Fukucho K, Kanazawa., Difference in Nutritive Value of a Microalgae Nannochloropsis oculata at Varous growth Phases. Bull Jap Soc Sci Fish. 56(8):1293-98. 1990

Toonen, R.J., Invertebrate Culture, J. Maquacluture 4(4): 6-25, 1996

Wilfors G, Ferris G, Smith B., The relationship between Gross Biochemical Composition of Cultured Algal Foods an growth of the Hard Clam Mercenaria mercenaria (L). Aquacluture 108(1): 135-154, 1992

Wilkerson, J., Clownfishes, Microcosm Limited; ISBN: 1890087041; June 1998.