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 Ethanol
Can 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 carbohydraterich 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.
2.
3.
4.
5.
6.
7.
8.
9.

Direct pyrolysis
Advanced mechanical extraction
Separation of sugar from biomass carbohydrate chains
Hybrid fuels
Low temperature fuels
Enzyme extraction
Algae strain development
High efficient LED artificial light production
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
info@GreenStarUSA.com. 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, President
619-864-4010
619-789-4743 fax
info@GreenStarUSA.com
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 CETANE GREEN

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 resourcesaving 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 fuelderived 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 highlytuned 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 310m. 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 emissions
Microalgae 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: 13.44

THE BREEDER'S NET by FRANK MARINI, Ph. D.

Sponsored in part by:

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
We are what we eat

Proud sponsor of this column

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

Algae

Total HUFA

EPA

DHA

N. Oculata

16-43%

High

Low

C. Gracilis

5-11.5%

0.3-2.5%

I. Galbana

2-4%

3-4.2%

0.2-0.7%

8.3-11%

~5%

~6%

T. Iso
Tetraselmis

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 511% 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

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.

maximum density. According to Wilkerson

[Wilkerson, 1998] this algae is too
temperamental, fragile, and fastidious to be used
regularly.
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.

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, of
importance is that there is no frothing or skimming on the
culture surface. Photo courtesy of Joe Burger.

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

Algae

Optimal pH

Temp Range

Minimum Illumination (LUX)

Salinity

N. Oculata

7.0-8.4

60-86

4,000-5,000

22-25

I. Galbana

7.8-8.5

77-86

1,000-6,000

28

Tetraselmis

6.9

68-82

1,000-20,000

30-40

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.

Proud sponsor of this column

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.

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 .

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, openended 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 overaerating 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 greenwater 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.com

Close 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 greenwater, 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 greenwater 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 greenwater 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: 504-736-9373
Aquatic 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 Phytoplanktons
Hamshire Il 60140
Phone 847-683-4564
(source for commercial N. Oculata)

Florida Aqua-Farms Inc

33418 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.htm
Provosoli-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.htm
http://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.