This
is the first in a series of microculture columns in which we’ll
explore the culture of larval fish foods. In this month’s column, we
focus on phytoplankton, the basic nutritional building block of our home
fish breeding effort. We’ll 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
Proud sponsor of this column
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 don’t 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%
T.
Iso
0.2-0.7%
8.3-11%
Tetraselmis
~5%
~6%
Nannochloropsis
oculata is a 2-4 micron (μm) 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 μm 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 μm 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, of
importance is that there is no frothing or skimming on the
culture surface. Photo courtesy of Joe Burger.
Tetraselmis
sp. is a 9-14 μm 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
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
Acommercially
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 ofthe 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
let’s 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 you’re 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 formulationis 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 you’ll
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 2-24”bulbs, 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 Burgerwww.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 let’s
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 don’t “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 don’t 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 you’re 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 you’ve 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) let’s 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 didn’t 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 I’ve
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
month’s 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 month’s column we’ll 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.
I’ll 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
Breeder’s Net.
Cripes,
D., Algae Nutrition, J MaquaCulture7(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.
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.
