Algal culture techniques

• The terminology used to describe the type of algal culture include:

• Indoor/Outdoor. Indoor culture allows control over illumination,

temperature, nutrient level, contamination with predators and

competing algae, whereas outdoor algal systems make it very difficult

to grow specific algal cultures for extended periods.

• Open/Closed. Open cultures such as uncovered ponds and tanks

(indoors or outdoors) are more readily contaminated than closed

culture vessels such as tubes, flasks, carboys, bags, etc.

• Batch, Continuous, and Semi-Continuous. These are the three basic

types of phytoplankton culture which will be described later

Batch culture
• The batch culture consists of a single inoculation of cells into a

container of fertilized seawater followed by a growing period of

several days and finally harvesting when the algal population reaches

its maximum or near-maximum density.

• In practice, algae are transferred to larger culture volumes prior to

reaching the stationary phase and the larger culture volumes are then

brought to a maximum density and harvested. The following

consecutive stages might be utilized: test tubes, 2 l flasks, 5 and 20 l

carboys, 160 l cylinders, 500 l indoor tanks, 5,000 l to 25,000 l outdoor

tanks
Production scheme for batch
culture of algae (Lee and
(.Tamaru, 1993
• Batch culture systems are widely applied because of their simplicity and

flexibility and often considered as the most reliable method,

• However, the quality of the harvested cells may be less predictable than

that of continuous systems and for example vary with the timing of the

harvest (time of the day, exact growth phase).

• Another disadvantage is the need to prevent contamination during the

initial inoculation and early growth period. Because the density of the

desired phytoplankton is low and the concentration of nutrients is high,

any contaminant with a faster growth rate is capable of outgrowing the

culture.

• Batch cultures also require a lot of labour to harvest, clean, sterilize, refill,

and inoculate the containers.

. Batch culture systems for the mass production of micro-algae in 20,000 l tanks
Batch culture systems for
the mass production of
micro-algae in 150 l
. cylinders
Carboy culture
apparatus
(Fox, 1983).
Carboy culture shelf (Fox, 1983).
 Nutritional value of micro-algae

• The nutritional value of any algal species for a particular

organism depends on its cell size, digestibility, production
of toxic compounds, and biochemical composition.

• Protein is always the major organic constituent, followed

usually by lipid and then by carbohydrate. Expressed as
percentage of dry weight, the range for the level of
protein, lipid, and carbohydrate are 12-35%, 7.2-23%, and
4.6-23%, respectively,,,,,note table 2.12 in page 32
 Nutritional value of micro-algae, cont….
• The content of highly unsaturated fatty acids (HUFA), in particular

eicosapentaenoic acid (20:5n-3, EPA), arachidonic acid (20:4n-6,

ARA), and docosahexaenoic acid (22:6n-3, DHA), is of major

importance in the evaluation of the nutritional composition of an

algal species to be used as food for marine organisms.

• Note in figure 2.14 of page 35,,,Significant concentrations of EPA

are present in the diatom species (Chaetoceros calcitrans, C.

gracilis, S. costatum, T. pseudonana) , whereas high concentrations

of DHA are found in the prymnesiophytes (Isochrysis sp.) and

Chroomonas salina.
Cymbella sp. after Nile red staining under fluorescence microscopy. Yellow
droplets show lipid containing droplets and orange droplets show chlorophyll.
 Use of micro-algae in aquaculture
• Bivalve molluscs
• Intensive rearing of bivalves has so far relied on the production of live algae,
which comprises on average 30% of the operating costs in a bivalve hatchery.
• the juveniles, representing the largest biomass in the hatchery and consume the
largest volumes of algal culture .
• Eight algal were reported in an international survey among hatchery (Isochrysis
sp., C. gracilis; C. calcitrans; T. suecica; T. pseudonana, clone 3H; P. lutheri; I.
galbana; S. costatum).
• The larvae of most bivalve species have similar food preferences; suitable algal
species including C. calcitrans, I. galbana, and T. suecica (for larvae > 120 μm in
length). Combinations of flagellates and diatoms provide a well balanced diet
which will generally accelerate the rate of larval development to metamorphosis
in comparison with unialgal diets.
• Penaeid shrimp

• Algae are added during the non-feeding nauplius stage so that algae are

available immediately upon molting into the protozoea stage.

• Algal species most often used are Tetraselmis chui, Chaetoceros gracilis, and

Skeletonema costatum.

• As feeding preference changes from primarily herbivorous to carnivorous

during the mysis stages, the quantity of algae is reduced.

• Marine fish

• algae are often used directly in the tanks for rearing marine fish larvae.

• This "green water technique" is part of the commonly applied techniques for

rearing larvae of gilthead sea bream Sparus aurata (50,000 cells ml-1 of

Isochrysis sp. + 400,000 cells.ml-1 of Chlorella sp. per day)

• The effects of the presence of micro-algae in the larval rearing tank are
still not fully understood and include:
• stabilizing the water quality in static rearing systems (remove metabolic by-
products, produce oxygen)
• a direct food source through active uptake by the larvae with the
polysaccharides present in the algal cell walls
• possibly stimulating the non-specific immune system in the larvae
• an indirect source of nutrients for fish larvae through the live feed (i.e. by
maintaining the nutritional value of the live prey organisms in the tank)
• increasing feeding frequency by enhancing visual contrast and light
dispersion, and microbial control by algal exudates in tank water and/or
larval gut.
 ROTIFERS
• Introduction
• Although Brachionus plicatilis was first identified as a pest in the pond culture of eels in the
fifties and sixties, Japanese researchers soon realized that this rotifer could be used as a
suitable live food organism for the early larval stages of marine fish.
• The successful use of rotifers in the commercial hatchery operations of the red sea bream
(Pagrus major) encouraged investigations in the development of mass culture techniques of
rotifers.
• The availability of large quantities of this live food source has contributed to the successful
hatchery production of more than 60 marine finfish species and 18 species of crustaceans.
• The success of rotifers as a culture organism are various, including their planktonic nature,
tolerance to a wide range of environmental conditions, high reproduction rate (0.7-1.4
offspring. female-1.day-1). Moreoever, their small size and slow swimming velocity make
them a suitable prey for fish larvae that have just resorbed their yolk sac but cannot yet
ingest the larger Artemia nauplii.
• However, the greatest potential for rotifer culture exist in in the possibility of rearing these
animals at very high densities (i.e. densities of 2000 animals.ml-1 have been reported by
Hirata (1979). Even at high densities, the animals reproduce rapidly and can thus contribute
to the build up of large quantities of live food in a very short period of time.
• Last, but not least, the filter-feeding nature of the rotifers facilitates the inclusion into their
body tissues of specific nutrients essential for the larval predators
• Morphology
• Males have reduced sizes and are less developed than females; some measuring
only 60 μm.
• The rotifer's body is differentiated into three distinct parts consisting of the head,
trunk and foot
• The head carries the rotatory organ or corona which is easily recognized by its
annular ciliation and which is at the origin of the name of the Rotatoria (bearing
wheels).
• The retractable corona assures locomotion and a whirling water movement which
facilitates the uptake of small food particles (mainly algae and detritus).
• The trunk contains the digestive tract, the excretory system and the genital
organs. A characteristic organ for the rotifers is the mastax (i.e. a calcified
apparatus in the mouth region), that is very effective in grinding ingested particles.
• The foot is a ring-type retractable structure without segmentation ending in one
or four toes.
 Biology and life history
• The life span of rotifers has been estimated to be between 3.4 to
4.4 days at 25o C.
• Generally, the larvae become adult after 0.5 to 1.5 days and
females thereafter start to lay eggs approximately every four
hours. It is believed that females can produce ten generations of
offspring before they eventually die.
• The reproduction activity of Brachionus depends on the
temperature of the environment
• The life cycle of Brachionus plicatilis can be closed by two modes of
reproduction:
• Parthenogenesis: the amictic females produce amictic (diploid, 2n
chromosomes) eggs which develop and hatch into amictic females.
• Under specific environmental conditions the females switch to a more
complicated sexual reproduction resulting in mictic and amictic
females. Although both are not distinguishable morphologically, the
mictic females produce haploid (n chromosomes) eggs. Larvae hatching
out of these unfertilized mictic eggs develop into haploid males. These
males are smaller in size; they have no digestive tract and no bladder
but have a single testis which is filled with sperm. Mictic eggs which will
hatch into males are significantly smaller in size, while the mictic
fertilized eggs (resting eggs) are larger and have a thick, weakly
granulated outer layer and hatch into amictic females after exposure to
specific environmental conditions.
• The resting eggs can be the result of changes in environmental
conditions eventually creating alternations in temperature or salinity or
changing food conditions.
 Strain differences
• Only a few rotifer species belonging to the
genus Brachionus are used in aquaculture;
Brachionus plicatilis is the most widely used.
Its a cosmopolitan inhabitant of inland saline
and coastal brackish waters.
• Two different strains , namely Brachionus
rotundiformis or small (S-type) rotifers and
Brachionus plicatilis or large (L-type) rotifers.
• They can be clearly distinguished by their
morphological characteristics: the lorica length
of the L-type ranging from 130 to 340 μm
(average 239 μm), and of the S-type ranging
from 100 to 210 μm (average 160 μm).
Moreover, the lorica of the S-type shows
pointed spines, while of the L-type has obtuse
(thick) angled spines.
 ……,, Strain differences cont
• In tropical aquaculture the SS-type rotifers (Super small rotifers, smaller
than S-strains) are preferred for the first feeding of fish larvae with small
mouth openings (rabbitfish, groupers, and other fish with mouth openings
at start feeding of less than 100 μm).
• The S- and L- morphotypes also differ in their optimal growth
temperature. The S-type has an optimal growth at 28-35°C, while the L-
type reaches its optimal growth at 18-25°C. Since contamination with
both types of rotifers occurs frequently, lowering or increasing culture
temperatures can be used to obtain pure cultures
 General culture conditions of marine rotifers
• Salinity
• Although Brachionus plicatilis can withstand a wide salinity range from 1 to 97
ppt, optimal reproduction can only take place at salinities below 35 ppt (Lubzens,
1987).
• However, if rotifers have to be fed to predators which are reared at a different
salinity (± 5 ppt), it is safe to acclimatize them as sudden salinity shocks might
inhibit the rotifers’ swimming or even cause their death.
• Temperature
• The choice of the optimal culture temperature for rearing rotifers depends on the
rotifer morphotype; L-strain rotifers being reared at lower temperatures than S-
type rotifers.
• In general, increasing the temperature usually results in an increased
reproductive activity. However, rearing rotifers at high temperature enhances the
cost for food and change water quality.
• At an expected point, high temperatures starving animals consume their lipid and
carbohydrate reserves very fast.
• Rearing rotifers below their optimal temperature slows down the population
growth.
• Dissolved oxygen
• Rotifers can survive in water containing as low as 2 mg.l/l of
dissolved oxygen.
• The level of dissolved oxygen in the culture water depends on
temperature, salinity, rotifer density, and the type of the food.
The aeration should not be too strong as to avoid physical
damage to the population.
• pH
• Rotifers live at pH-levels above 6.6
• Ammonia (NH3)
• The NH3/NH4 ratio is influenced by the temperature and the
pH of the water.
• High levels of un-ionized ammonia are toxic for rotifers but
rearing conditions with NH3-concentrations below 1 mg/l
appear to be safe.
• Bacteria
• Pseudomonas and Acinetobacter are common opportunistic bacteria which
may be important additional food sources for rotifers. Some Pseudomonas
species, for instance, synthesize vitamin B12 which can be a limiting factor
under culture conditions.
• Although most bacteria are not pathogenic for rotifers their proliferation
should be avoided since the real risk of accumulation and transfer via the food
chain can cause harmful effects on the predator
• Some pathogenic bacteria like Vibrio anguillarum which is common in culrure
may causing a negative effect on rotifers cultured on a sub-optimal diet while
the rotifers grown on an optimal diet were not affected by the bacterial strain.
• Ciliates
• Holotricha and Hypotricha ciliates, such as Uronema sp. and Euplotes sp., are
not desired in intensive cultures since they compete for feed with the rotifers.
• Ciliates produce metabolic wastes which increase the NO2-N level in the water
and cause a decrease in pH. However, they have a positive effect in clearing
the culture tank from bacteria and detritus.
• The addition of a low formalin concentration of 20 mg.l-1 to the algal culture
tank, 24 h before rotifer inoculation can significantly reduce protozoan
contamination.
 Stock culture of rotifers
• Culturing large volumes of rotifers
on algae, baker's yeast or artificial
diets always involves some risks for
sudden mortality of the population.
• Technical or human failures but
also contamination with pathogens
or competitive filter feeders are the
main causes for lower reproduction
which can eventually result in a
complete crash of the population.
Stock cultures of rotifers kept in 50 ml
• Small stock cultures are generally centrifuge tubes. The tubes are fixed on a
kept in closed vials in an isolated rotor. At each rotation the medium is
room to prevent contamination mixed with the enclosed air.
with bacteria and/or ciliates. These
stock cultures which need to Read pages 56&57 for
generate large populations of preparation of stock culture
rotifers as fast as possible are
generally maintained on algae. Up scaling of stock cultures to starter cultures,,
pages 57 & 58
 Mass production on algae
• Undoubtedly, marine microalgae are the best diet for rotifers
and very high yields can be obtained if sufficient algae are
available and an appropriate management is followed.
• Unfortunately in most places it is not possible to cope with the
fast filtration capacity of the rotifers which require continuous
algal blooms
• however, pure algae are only given for starting up rotifer cultures
or to enrich rotifers.
• Batch cultivation is probably the most common method of rotifer
production in marine fish hatcheries. The culture strategy
consists of either the maintenance of a constant culture volume
with an increasing rotifer density or the maintenance of a
constant rotifer density by increasing the culture volume
 Mass production on algae and yeast
• Depending on the strategy and the quality of the
algal blooms baker's yeast may be supplemented.
The amount of yeast fed on a daily basis is about 1
g.million/l of rotifers
• Since algae have a high nutritional value, an
excellent buoyancy and do not pollute the water,
they are used as much as possible, not only as a
rotifer food, but also as water conditioners and
bacteriostatic agents.
• The mass production on algae and yeast is
performed in a batch or semi-continuous culture
system.
 Mass culture on yeast
• Baker's yeast has a small particle size (5-7 μm) and a
high protein content and is an acceptable diet for
Brachionus, however The occurrence of sudden
collapses of the cultures is frequently occur.
• Most probably the reason for these crashes was
explained by the poor digestibility of the yeast,
which requires the presence of bacteria for
digestion.
• Moreover, the yeast usually needs to be
supplemented with essential fatty acids and
vitamins to suit the larval requirements of the
predator organisms.
 Nutritional value of cultured rotifers
• Techniques for (n-3) HUFA enrichment
• Algae
• The high content of the essential fatty acid
eicosapentaenoic acid (EPA 20:5n-3) and
docosahexaenoic acid (DHA 22:6n-3) in some microalgae
(e.g. 20:5n-3 in Nannochloropsis occulata and 22:6n-3 in
Isochrysis galbana) have made them excellent live food
diets for improving the fatty acid content of the rotifers.
• Rotifers submerged in these algae (approximately 5.106
algae.ml/l) are incorporating the essential fatty acids in a
few hours
• However, the culture of microalgae as a sole diet for
rotifer feeding is costly due to the labour
• Oil emulsions
• One of the cheapest ways to enrich rotifers is by using oil emulsions.
• Home-made emulsions
• The first emulsions were made from (n-3) HUFA rich fish oils (i.e.
cuttlefish oil, Pollack liver oil, cod liver oil, etc.) and emulsified with egg
yolk and seawater (Watanabe et al., 1982, 1983). Recently, more
purified oils containing specifically high levels of the essential fatty
acids 20:5n-3 and 22:6n-3 have been used.
• Commercial emulsions
• Several combined diets are commercially available and based on well-
defined formulations. Very popular are the self-emulsifying
concentrates (Selco®, Inve Aquaculture NV, Belgium) which can
enhancement the HUFA content of the rotifers in a few hours.
• In this technique a rotifer suspension containing 200-300
individuals.ml-1 is immersed in a diluted oil-emulsion for 6 h,
harvested, rinsed and concentrated before being fed to the predators.
• Techniques for vitamin C
enrichment
• The vitamin C content of rotifers reflects
the dietary ascorbic acid (AA) levels both
after culture and enrichment
• For example, rotifers cultured on instant
baker’s yeast contain 150 mg vitamin C/g-
1 DW, while for Chlorella-fed rotifers
contain 2300 mg vitamin C/g-1 DW.
• Enrichment of rotifers with AA is carried
out using ascorbyl palmitate (AP) as a
source of vitamin C. AP is converted by the
rotifers into active AA up to 1700 mg.g-1
DW after 24 h enrichment using a 5 % AP .