Many bivalve hatcheries increase larval production by including an algal
culture operation within the facility. Vats of dense algae provide mollusc
larvae with a nutritious diet. The high quality diet accelerates growth and
shortens the time to larval settlement or spat set. Production of algae in large
amounts (mass cultures) is accomplished by providing a favorable environment for
the species being cultured. Successful algal culture follows six basic
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Algal culture begins with a pure stock or starter culture of the algal
species desired. These can be obtained from a number of sources. Commercial
biological supply houses often sell algal cultures, but the species may not be
suitable for use in your operation. Stock cultures of various species are often
kept at government laboratories and universities. Perhaps the most common method
of obtaining algae is to visit a working hatchery, since many hatchery operators
will be glad to provide starter cultures to other culturists. Sources of algal
species and culture supplies are listed near the end of this fact sheet.
Once obtained, starter cultures (usually transported in test tubes) are used
to inoculate several new cultures. Some of these are kept as stocks for when an
old culture dies, is harvested, or otherwise lost and must be restarted. The
rest are used to inoculate progressively larger vessels until there is enough
culture to start mass production tanks. The culturist must supply
light, aeration, relatively stable temperature control, and sterile water with
nutrients (media) to produce successful algal cultures. By avoiding major
contamination from foreign algal species and microscopic predators, a
continuous, dependable supply of high quality algae will be available.
The information in this guide can be used by hatchery operators to produce
algal food for various bivalve organisms such as oysters, clams, scallops, or
Bivalves grow and develop primarily by consuming microscopic plants. These
extremely small, free-floating, single-celled (unicellular) plants are called
phytoplankton or microalgae. Individually, the plants are 2-10 micrometers (um)
or about 0.00008-0.00040 inches in size. They are invisible to the unaided eye.
Their presence is apparent only when thousands of the algal cells come in close
contact with each other and color the water green, red, or golden brown. A
microscope with magnification of 400 power (400x) reveals their shape, size,
abundance, and movement.
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There are thousands of microalgae that differ in size, color, shape, and
habitat. Researchers have isolated and identified several species from natural
waters that have proven to be nutritious food for growing some commercially
important bivalves. Pavlova (Monochrysis), Dicrateria, Thalassiosira,
Isochrysis, and Isochrysis (T-Iso strain) are used by personnel at the Frank M.
Flowers & Sons Hatchery in Bayville, NY. Culturists with Ocean Pond
Corporation in Fishers Island, NY use predominantly Isochrysis (T-Iso strain).
Other species commonly used include Chaetoceros calcitrans, Thalassiosira
weissflogii, Dunaliella tertiolecta, Nannochloris aromus, Tetraselmis suecia,
and some species of Chlorella. The University of Maryland's Horn Point oyster
hatchery in Cambridge, MD has had success feeding Isochrysis galbana (T-Iso
strain) and Thalassiosira pseudonana (strain 3h) to oysters and several species
of clams. I. galbana is a five um (micrometer) golden brown, spherical cell. It
possesses two hair-like flagella at its anterior end for motility. T. pseudonana
is a 4 um, non-motile, golden brown, barrel-shaped cell.
Under favorable conditions, unicellular algae grow continuously by a process
known as cell division. Each cell enlarges and divides into two daughter cells
that subsequently grow and divide yielding a culture that increases
exponentially (e.g., 8, 16, 32, 64...etc.).
Growth slows as the algal population becomes more crowded. Nutrients are
depleted, metabolites build, and light penetration decreases because of
self-shading. The cultures have reached their stationary phase for
the current conditions and will not increase in density. Algae harvested near
this maximum density are a high quality food and provide the most efficient use
of hatchery labor and space.
The amount of time for algae to divide and the maximum density attained
depend upon several factors:
Growth of unicellular algae can be measured in terms of cell numbers in a
given volume of water. In natural waters, algal density rarely exceeds a few
thousand cells per milliliter (one mL is equal to about 20 drops from an eye
dropper). In a controlled hatchery environment, however, a single algal species
or "monoculture" of Isochrysis galbana can grow from 200,000 cells/mL to four
million cells/mL in three days.
The first step in algal culture is the preparation of growth media. Media are
clean, nutrient enriched, sterilized solutions of estuarine or sea water. The
water should be approximately the same salinity as the water in which the
bivalves are grown. Water collected from a healthy shellfish nursery area should
provide adequate media for growing algae; However, aged water (one week or more)
or water from a salt water well are superior for algal culture compared to
freshly collected estuarine water. It is generally not practical to store large
volumes of water, but a small supply for stock cultures can be kept on hand.
To make media, water clouded with silt and debris should first be cleared by
filtering or settling in a holding tank, preferably in the dark, for several
days. Filter pore size is determined by the amount and size of the debris in the
water column. For example, polypropylene cartridge filters with a one or two um
pore size are required to remove small suspended clay particles. Hatchery
operators at many locations may find such fine filtering unnecessary. For
instance, the F. M. Flower's hatchery pumps silt-free, aged water from a salt
Once collected, the water should be sterilized. Small amounts (one pint or
less) of saline water are sterilized in heat-resistant, loosely covered
containers. A pressure cooker or autoclave is commonly used with a sustained
temperature of 121' C (250' F) at 15 p.s.i. for 15 minutes. An alternative
method is to boil the container, including the seawater, either in a double
boiler or directly on a burner for half an hour. Either method effectively kills
all living organisms present and destroys spores or resting stages. Some
culturists have used microwave ovens to successfully sterilize small amounts of
water. Other culturists sterilize saline water by passing it through a filter
with a 0.45 um or smaller pore size into a sterile vessel. These small,
sterilized containers and waters are used for stock cultures.
Boiling saline water may cause precipitates to form which inhibit algal
growth. This phenomenon is usually more prevalent in higher salinity water (130
parts per thousand). A better method uses heat pasteurization to kill unwanted
microorganisms. The saline water is heated to 80'C (176'F) and allowed to cool
naturally to room temperature. Spores can be killed by heating and cooling the
water a second time. If the water is not used soon after cooling care should be
taken to insure against contamination. Hatcheries in locations where heavy silt
loads are not a problem may use a filtration system to sterilize water
mechanically. Filters with an absolute pore size of one or two um should be
used. Filter sterilization could eliminate heating and chemical costs.
A more practical sterilizing technique for large volumes (one gallon or more)
of saline water requires the use of common chemicals. Either laundry bleach
(sodium hypochlorite) at 0.5 milliliters per liter (mLs/L) of culture water or
hydrochloric acid, also called muriatic acid, can be used (Table 1). Once
combined, the water and chemical mixture is left for 6-24 hours. Treatment with
bleach or acid renders the water "clean" enough for an algal inoculum.
The acid is deactivated with sodium bicarbonate (baking soda; Table 2). The
bleach is deactivated with 0.10.15 mLs (for each liter of chlorinated water) of
a sodium thiosulfate (248 grams/L) solution and aerated for two or more hours.
The presence of chlorine should be checked. Inexpensive test kits are available
from pool supply stores.
Concentrated Acid Added
Sodium Bicarbonate Added to Cultures
Acid sterilization lowers the pH of water to 3.6 or less. Occasionally a
bicarbonate residue is left on the walls of the vessel from a previous culture.
This requires an increase in the amount of acid, and a check with a pH meter to
ensure the desired pH is attained. To avoid contamination, the treated water and
interior of the container must not contact anything except the seed algae and
After sterilization, the water is enriched to provide the cultured algae with
nutrients needed for rapid growth to high densities. There are numerous recipes
available for the preparation of algal nutrients. The one used depends on
nutrient levels in local, ambient water and the algal species cultured. Many
algae grow well with Guillard's f/2 nutrients.
Guillard's f/2 nutrients are used at the rate of 2 mLs for each liter of algae
cultured. The trace metal and vitamin solutions are saved to spike 10 batches of
primary f/2 solution as needed. Guillard's f/2 and other nutrients can also be
purchased ready-to-use from suppliers listed near the end of this guide.
Experienced culturists can modify a recipe to increase growth or reduce cost.
For example, vitamins, an expensive ingredient, can sometimes be omitted from
mass tank cultures. Some hatcheries have used off-the-shelf fertilizer, a
practice that may not produce consistently reliable growth. Fertilizers should
be compared and those containing high concentrations of heavy metals should be
avoided. Diatoms, including T. pseudonana, are enhanced in some locations with
the addition of 0.03 grams of sodium silicate per liter of media. Typically a
separate solution (30 grams/L) of sodium silicate is mixed and added to diatom
cultures (15 mLs/S gallons). I. galbana mass cultures grow well on Stern's
"Miracid" (plant food) alone, used at the rate of 1.3 oz. per 240 gallons (0.04
The algal inoculum or seed is added soon after the nutrients and chemical
neutralizer. The algae are not harmed even though the baking soda will not be
completely dissolved for several hours. Ordinarily enough algae are added to
produce a strong tint in the water (100,000-200,000 cells/mL). More inoculum is
used if peak density is desired quicker than usual. The culture is illuminated
and may be aerated.
Sunlight, fluorescent, VHO fluorescent, and metal halide. lights can be used
either singly or in various combinations to provide light. Special "plant grow"
lights do not increase algal production. A tank eight feet long, four feet wide,
and one foot deep can accommodate 16, eight foot fluorescent tubes, and yield
dense cultures. Several smaller cultures in pint, quart, or five gallon bottles
can be placed in front of four-foot, two or four bulb (40 watt each) fluorescent
tubes. Generally, the more light the greater the final peak algal densities.
Decreasing the depth of algae in a mass tank culture can have the same effect as
higher light intensity. Low density cultures, such as fresh inoculations of I.
galbana , (less than approximately 50,000 cells/ mL) require much lower light
levels initially. This can be accomplished by using window screening to shade
some sunlight, by turning off some bulbs for the first 24 hours, or increasing
the distance between the tubes and the cultures. Fluorescent 2 bulb light
fixtures are commercially available in various lengths (2, 4, and 8 feet). They
can be purchased according to the size tanks that are to be illuminated.
Stock cultures are kept on hand for initial start-up and as a source of
"clean" algae when the mass tank cultures or the smaller intermediate cultures
become contaminated, die, or are harvested.
Stock cultures are usually kept in heat-resistant, transparent test tubes or
conical flasks. Flasks are available in sizes ranging from 1.7 fl. oz. to 4
quarts (50 to 4,000 mLs). The culturist can choose the size that is most
convenient and practical for his operation. An adequate volume (usually 250 mLs
or more) of stock culture must be available for the next step in vessel size.
Air is not bubbled into stock flasks, thus eliminating one potential source of
contamination. Each flask is half filled (or less) with media. Partially filled
flasks provide a larger surface area for gas exchange than full flasks. Good gas
exchange increases the prospect of stable, long-lived, and healthy stock
Many hatcheries keep back-up stock cultures. Some are kept on-site in
duplicate culture chambers. Others are kept at locations remote from the
hatchery. In this way problems with equipment, power failures, unexplained
crashes, and other unforeseen difficulties can be minimized.
By following a strict transfer regime, pure algal monocultures can be kept
healthy and used.indefinitely to seed new cultures. For example, once a week,
one drop of T. pseudonana inoculant is aseptically transferred to media
in a newly sterilized, I-liter flask. A I. galbana inoculant is typically
transferred once every two weeks. The amount transferred and frequency depends
on the needs of the hatchery.
Stock cultures are used to inoculate intermediate-sized cultures. Many
hatcheries use five gallon bottle (19 L) cultures as a step between flask stock
cultures and large mass tank cultures. These bottles are available in either
plastic (polycarbonate) or glass. Glass bottles are heavy and breakable, but can
be autoclaved without ill effects. Light-weight plastic bottles have seams that
could develop leaks, particularly if autoclaved. Other intermediate-sized
vessels such as translucent 2.5 gallon bottles, plastic one gallon milk jugs or
polyethylene bags can be used. Polyethylene material for constructing bags can
be purchased as tubing (diameters from 1.5 - 58 inches). The ends can be heat
sealed to any convenient length. They require external support, but are
disposable and don't need to be sterilized when first used. Many hatcheries are
using this technique to mass culture algae.
For a small, laboratory-sized hatchery, these containers may provide enough
algae to satisfy production needs. Commercial-size bivalve hatcheries require
mass tank cultures containing 25-5,000 gallons (100-20,000 L) or more of quality
algae. In these operations, mass tank cultures are typically started from
healthy bottle cultures.
Tank and bottle cultures should be provided with the most favorable
environment possible. Deviating from ideal conditions will reduce algal growth
rate. Lower light levels or reliance on natural sunlight could be used to reduce
electrical costs, but with a concurrent decrease in production. Some hatcheries
eliminate aeration to reduce the spread of biological contaminants. Departures
from optimal conditions may be necessary for other reasons. For example, if
several algal species are cultured in one room, a compromise in environmental
conditions may be necessary. A cooler temperature, such as 65' F, would increase
the time needed to attain peak density for some species, but may eliminate a
Inoculum sufficient for a bottle produces a visible tint in the water. For
example, one or two pints (500-1,000 mLs) would be an adequate inoculum for a
five gallon bottle. Under favorable conditions, a newly inoculated I.
galbana culture in a five gallon (19 L) bottle takes 10-14 days to reach
peak density (10-12 million cells/mL). The faster growing T. pseudonana
reaches its peak (four million/mL) in about three days. Once maximum density
is attained, healthy I. galbana bottle cultures are stable for two
additional weeks. Cultures of T. pseudonana will deteriorate within five
days once reaching maximum density. Growth rate and culture stability of most
other algae fall between these two.
Using a larger volume of inoculum will decrease the time for a culture to
reach peak density. For example, a half gallon (2 L ) inoculum instead of one
pint (500 mLs) would shorten by 30-50% the time a five gallon (19 L) bottle
needs to reach peak density. Algal growth could be increased by bubbling in a
mixture of air and 10% carbon dioxide instead of air alone. Fast growing
cultures could be enhanced with carbon dioxide pulsed in several times a day,
keeping the pH below 10. Carbon dioxide is available in pressurized bottles from
bottled air suppliers in most communities.
A dense (10 million cells/mL), healthy five gallon (19 L) bottle Culture
contains enough inoculum to seed a 240 gallon (900 L) mass tank culture. At
lower densities (five million cells/mL, for example), two or more bottles would
be necessary to produce the same amount of algal inoculum. Larger tank cultures
would benefit from a proportionally larger inoculum. Mass cultures in 240 gallon
tanks (8'x 4'x 1') should reach peak density in 2-3 days when configured as
compares the general growth parameters for I. galbana and T.
pseudonana. The mass tank cultures should be harvested near peak density and
before biological contamination becomes a problem. Some contaminants, as
explained in a later section, will quickly destroy the algae.
Two algal harvesting methods can be used: 1) batch and 2) semi-continuous.
With the batch method, a total harvest occurs at once or over several days. The
tank or bottle is then cleaned, refilled and prepared for a new inoculum. In the
semi-continuous method, part of the algae remains in the vessel and serves as an
inoculum. New media is added to replenish the amount of algal suspension
removed. The semi-continuous technique works well with diatoms (T. pseudonana,
Chaetoceros spp.). Diatoms outgrow foreign algae and many other biological
contaminants. The most appropriate harvest method will depend on the water
quality and the algal species cultured. A salt water well typically provides the
best media for the semicontinuous method.
Monitoring algal growth in mass tank cultures is essential for successful
production. Cultures that have stopped growing are either:
1) significantly contaminated with microscopic predators and/or
competitors, or 2) have reached maximum ceil density under the present
1) significantly contaminated with microscopic predators and/or
competitors, or 2) have reached maximum ceil density under the present
A general gauge of algal growth is the daily darkening color as the cells
multiply. A more accurate measure of growth can be made by comparing actual cell
numbers with the previous day's count.
The number of cells can be determined by studying a sample from a culture
under a 400X microscope. The sample is placed on a special slide called a
hemacytometer. These slides are available from scientific or medical supply
stores and come with instructions. To use the hemacytometer, place a pipet
filled with a sample from the culture tank against the "V" groove of the slide
(a cover slip must be in place). Withdraw the pipet before the sample runs off
the flat grid area, but not before the grid area is completely covered with the
algal suspension. Wait about five minutes for the cells to drift onto the grid.
The grid area resembles the pound symbol (#) inside a square border so that
there are nine equal squares. Each of the four corner squares are further
divided into 16 smaller squares. The total number of algal cells in all 16 small
squares in the four corner areas (64 total small sqs.) are counted. Divide this
number by four, for an average, and multiply by 10,000 (hemacytometer volume
factor). The product is equal to the number of cells per mL in the sample.
Motile algae like I. galbana, must be killed before counting. A weak toxin such
as acetic acid or ethanol may be used (one drop into 20-30 mLs of algal sample).
Dense cultures (>3 million) should be diluted 1:10 to facilitate counting and
to reduce error. Remember to multiply by 10 (for a 10:l dilution) or by the
appropriate correction factor if a different dilution is made. There are
numerous other methods to quantify algal densities, however using a microscope
and hemacytometer, will reveal the presence of biological contaminants.
Peak or maximum density (for existing conditions) is attained when, on
consecutive days, the cell count does not increase by a factor of two (Figure
2). With practice, approximate cell counts can be estimated by observing the
gross color of each culture. These estimates are based on the tint of the water
caused by the algal cells and comparing it to the actual cell count. Tedious and
time consuming cell counts (or other growth measurements) can be eliminated from
the daily routine. Cell counts should be made periodically as a check on the
As the cultures reach their peak density and are used, new cultures are
inoculated as replacements. A total of 15 bottles (five gallon) of I. galbana
cultured over three weeks are needed to grow five million oyster larvae from
eggs to "setting." This assumes a peak algal density of 10-12 million cells/mL
and a larval density of 5/mL. Many factors including food, salinity, brood
stock, disease, and water quality affect the growth and survival of each
Ideally, larvae are provided with a continuous supply of cultured microalgae
until they are ready for setting. (At setting the bivalve larvae metamorphose
into the adult form.) Feeding levels should be adjusted according to the density
and grazing activity of larvae. Larvae (5-10 organisms/mL) up to one week old,
grow well when initial algal concentrations in the tank proximate 50,000 cells/
mL. Doubling or halving this density during week one produces no discernible
growth differences. A daily adjustment to approximately 100,000 algal cells/mL
for larvae over one week old is adequate. Near the onset of setting, feed
requirements of bivalve larvae increase. Algal densities are raised to 200,000
cells/mL. Higher algal densities do not increase growth and may be detrimental
to the larvae. A mixture of several algal species is frequently used to enhance
growth. Generally, sufficient algae is added so the tank will not be completely
cleared in 24 hours.
Algal concentrations in the larval tanks can be monitored with a
hemacytometer as described above. With experience, feeding can be accomplished
by the visual or "eyeball" method (observance of the water tint caused by the
addition of algae). Novice culturists can use the following formula as a guide
to determine the volume of algae (5 um algal cell size) to add:
Liters to feed = (TD x V) / CD
Liters to feed = (TD x V) / CD
Where TD equals the algal Target Density in the larval tank (in thousands of
cells/mL); V is the Volume of the larval tank (in thousands of L); CD is the
Cell Density of the algae feed tank (in millions of cells/mL).
Example 1. To feed a population of larvae 50,000 cells/mL in a 6,000 liter
tank from an algal culture with 3.5 million cells/mL would require how many
liters of algae?
(50 cells/mL x 6 L) / 3.5 cells/mL = 86 L
(50 cells/mL x 6 L) / 3.5 cells/mL = 86 L
Example 2. To feed larvae at the rate of 200,000 cells/ mL in an 8 L
container from an algal culture with 12 million cells/mL would require how much
(200 cells/mL x 0.008 L) / 12 cells/mL = 0.133 L
Contamination of cultures by live microscopic predators and competitors is a
problem that cannot always be controlled successfully. The 2.5-5 gallon bottles
and small stock cultures can be isolated from live, airborne contaminants (such
as alien algae, bacteria, and predatory protozoans). A cotton plug, aluminum
foil, plastic wrap, or a screw cap can be used to seal the top. The large, open
mass tank cultures cannot be covered as readily as small- mouth containers. Mass
tank cultures are easily contaminated from non-sterile containers or splashed
water from buckets, hoses, or workers hands. Another source of contamination may
be in the media itself. Chemical sterilization does not kill the resting or
spore stage of all microscopic organisms. When the media is initially readied
for the algae, the environment also becomes favorable for the hardy spores of
any organism that is present. They begin to proliferate along with the
inoculated algae. For this reason autoclaved water is the preferred sterilizing
technique for stock cultures.
A quick microscopic examination of suspect, "different looking" cultures
enables the culturist to detect biological contaminants. The goal is to
determine if an algal die-off (crash) is likely. Knowing the status of the
growing algae helps the culturist to decide when the next mass culture should be
started. Common contaminants include unwanted algal species, bacteria, and
protozoans. Many flagellated protozoans can have a devastating effect on an
algal culture. They engulf and consume individual algal cells, while their
population grows exponentially (eg., 2, 4, 8, 16, ... etc.), similar to
An algal culture that is contaminated with certain species of protozoans
(such as Monas spp.) is likely to be completely destroyed within 12 to 18 hours
of first detection. Diligent monitoring of the culture with a 400 power
microscope is the only way to be certain of its presence. Even with 90% of the
algal cells destroyed, the culture can retain a brown tint similar to a healthy
algal culture. An algal "crash" caused by this protozoa renders the culture
useless as larval food. If inadvertently used as mollusc food, the larval growth
rate will be severely hindered. Not only are the protozoa very efficient at
eliminating the algae, they also offer little or no nutritional value to the
larvae. Algal cultures heavily infested by Monas are best dumped.
Although T. pseudonana cells are attacked by these protozoans, they
are not as susceptible as I. galbana cells. An algal culture with a protozoan
invasion should be discarded or used as larval food only as a last resort.
Inoculating a new culture with a protozoan-contaminated culture is
The Monas culprit is identified by its pale color, larger size (11 um,
spherical, or pear shape), and slower, sometimes circular movement. One or more
algal cells can usually be seen within its cell membrane.
Cultures that become contaminated with foreign algae, bacteria, or organisms
other than protozoans do not usually suffer a complete algal "crash." These
cultures grow slower and reach a lower final density than that of pure
monocultures. Such cultures should not be used as an inoculum, but are suitable
as larval food with a somewhat reduced nutritional value. Slow growing foreign
algae, causing minor contamination, can generally be ignored. Cultures that
become overrun with blue-green algae, which offer little nutrition to immature
molluscs, should be discarded to make room for new, pure cultures. Diatoms and
dinoflagellates larger than about 25 um are too large for most bivalve larvae to
Bacteria, present in small numbers, are unavoidable and normally do not
adversely affect the algae or larvae. Ingested bacteria probably play a positive
role in the nutrition of the larvae. Large bacterial populations are associated
with senescent (old, non-growing) algal cultures. Two methods are effective in
avoiding senescent cultures: 1) adding fresh media to the culture or 2)
transferring some of the algal solution to new media in a separate, sterile
A vigorous effort to avoid contamination should be the top priority of the
mass algal operation. This is accomplished by keeping all equipment meticulously
clean (chemically and biologically) and by avoiding introduction of any foreign
substance into the cultures. The rule is: start with a pure inoculum, grow the
algae under the most ideal conditions possible, and use the algae before
biological contaminants become established. Early detection of protozoans by
microscopic examination enables the culturist to avoid the unwitting use of
Summary of Steps to Culture Microalgae
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The basic equipment and resources required to culture microalgae successfully
The authors wish to thank David Relyea for his comments and suggestions, and
Joseph Buttner for arranging review of the manuscript. This work was supported
by the Northeastern Regional Aquaculture Center through grants number
89-385004356 and 90-38500-5211 from the Cooperative State Research Service, U.S.
Department of Agriculture. Any opinions, findings, or recommendations expressed
in this paper are those of the authors and do not necessarily reflect the views
of the U.S. Department of Agriculture.
Dupuy, J.L., N.T. Windsor and C.E. Sutton. 1977. Manual for Design and
Operation of an Oyster Seed Hatchery for the American Oyster, Special Report No.
142 in Applied Marine Science and Ocean Engineering of the Virginia Institute of
Marine Science, Gloucester Point, Virginia.
Smith, L. D. 1977. A Guide to Marine Coastal Plankton and Marine Invertebrate
Larvae. Kendall/Hunt Publishing Company, Debuque, Iowa.
Smith, W.L. and M.H. Chanley (eds.). 1975. Culture of Marine Invertebrate
Animals. Plenum Press, New York.
Stein, J.R.. 1973 Handbook of Phycological Methods, Cambridge University
Press, Cambridge, England.
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