FI:DP/CPR/81/014 Field Document 2 June 1988

PEOPLE'S REPUBLIC OF CHINA

prepared for the project
Development of marine culture of fish

by

Pinij Kungvankij
Consultant (Hatchery Management)

This report was prepared during the course of the project identified on the title page. The conclusions and recommendations given in the report are those considered appropriate at the time of its preparation. They may be modified in the light of further knowledge gained at subsequent stages of the project.

The designations employed and the presentation of the material in this document do not imply the expression of any opinion whatsoever on the part of the United Nations or the Food and Agriculture Organization of the United Nations concerning the legal or constitutional status of any country, territory or sea area, or concerning the delimitation of frontiers.

FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS
Rome, 1988

Hyperlinks to non-FAO Internet sites do not imply any official endorsement of or responsibility for the opinions, ideas, data or products presented at these locations, or guarantee the validity of the information provided. The sole purpose of links to non-FAO sites is to indicate further information available on related topics.

This electronic document has been scanned using optical character recognition (OCR) software. FAO declines all responsibility for any discrepancies that may exist between the present document and its original printed version.

TABLE OF CONTENTS

1. INTRODUCTION

1.1 Terms of Reference
1.2 Background Information

2. SELECTION OF FOOD

3. PHYTOPLANKTON CULTURE

3.1 Types of Algal Cultures
3.2 Sampling/Isolation Techniques
3.3 Growth
3.4 Conditions Affecting Growth

3.4.1 Illumination
3.4.2 Temperature
3.4.3 Culture Medium
3.4.4 Starter or Inoculum

Appendix 1: Culture Media and their Preparation

Appendix 2: Nutrient Media Utilized for Various Phytoplankton Species in SEAFDEC Phycology Laboratory

LIST OF FIGURES

1. The characteristic pattern of growth shown by unicellular algae in a culture of limited volume

2. Programming of natural food culture for hatchery operations

3. Schematic diagram of the initial culture method for rotifers, called “daily tank-transfer”

4. Conceptional diagram of mass-culture of rotifers in a “drain-off” system in large outdoor tanks

1. INTRODUCTION

1.1 TERMS OF REFERENCE

The Government of the People's Republic of China, assisted by the United Nations Development Programme and the Food and Agriculture Organization of the United Nations, is engaged in the project Development of marine culture of fish (CPR/81/014), whose main objectives are to develop the culture techniques including seed production of marine finfish in the coastal areas of China.

As part of the project operations, FAO assigned Mr Pinij Kungvankij as hatchery management consultant from 10 to 23 January 1988 and 29 January to 12 February 1988. During the course of his assignment, the consultant prepared this Guide.

1.2 BACKGROUND INFORMATION

To date, mass-rearing of fish larvae is still the most problematic aspect of the artificial propagation of marine finfish. One of the most important factors affecting the successful massive rearing of fish larvae is a suitable feed supply.

Generally, the eggs of brackishwater and marine finfish are small, and consequently the larvae are also small and fragile. It is not yet feasible to start feeding them with artificial diet in mass-culture systems. They have to be fed live food that can be easily mass-produced in the hatchery. The most commonly used live foods include: rotifer, mollusc larvae, phytoplankton and brine shrimp nauplii.

2. SELECTION OF FOOD

When selecting food to be fed to the larvae the following points should be considered:

1. The food must be perceived by the larvae. From this point of view, live food is preferred to inanimate food during the first few days of feeding.

2. The size of food must be such that it can be accommodated by the mouth of the larvae.

3. The feed should have high dietary value especially Highly Unsaturated Fatty Acids (HUFA) essential to the growth and survival of the larvae.

4. The feed can be easily produced in large quantities.

5. The feed can be digested by the larvae.

3. PHYTOPLANKTON CULTURE

In the past rearing methods employed in most hatcheries involved the direct fertilization of the tank water which contained natural populations of phytoplankton. It was observed, however, that this method often resulted in heavy blooms not only of plankton species of the desired size and kind, but also of others with the result that algal densities in the tanks proved difficult or even impossible to maintain at optimum levels.

The general practice in larviculture is to use mixed diets consisting of phytoplankton feed during the early larval stages and zooplankton for the successive stages of growth. The phytoplankters play vital roles in the food chain for they serve as food not only for the larvae but also for zooplankton.

In larviculture, the phytoplankton may serve any of the following functions: (1) provide nutrients via accidental or active ingestion by the larvae; (2) detoxify the larviculture medium by assimilating or neutralizing inhibitory material; (3) improve the nutritional value of secondary food organisms such as zooplankton; and (4) secrete into the medim metabolic products which facilitate larval growth and/or development.

Therefore, a knowledge of the abundance of these natural food organisms, their reactions, requirements and chemical composition is essential for better understanding of the nutritional requirements of whatever species is under review.

3.1 TYPES OF ALGAL CULTURES

The various types of algal cultures include :

• Maintenance cultures - natural collections of algae kept in culture vessels in the laboratory; here, succession of the previously less abundant species over the dominant ones may occur.

• Enrichment cultures - refer to crude collections of algae or other algal source materials treated with specially selected culture media which will favour the rapid increase in number of desired algal species.

• Unialgal cultures - refer to populations consisting of a single algal species, although other micro-organisms may be associated.

• Axenic culture - contains a population of a single algal species, all other living organisms being absent.

3.2 SAMPLING/ISOLATION TECHNIQUES

Minute-sized algae, particularly the unicellular forms, have submitted to the same techniques that are presently being employed for bacteria, protozoa, yeasts and moulds.

Phytoplankton may be collected by towing through the water special plankton nets made of fine silk bolting cloth (180 meshes/inch²). A small vial is attached to the end of the net which serves as a collector. Collected samples may also be centrifuged or filtered. Samples for examination may be preserved in 10–15% formalin.

Sampling bottles should be uncovered and illuminated promptly upon reaching the laboratory. It should be noted that a natural collection kept in the laboratory immediately begins to alter its state and composition. Competition among species is one of the main difficulties in maintaining a mixed population. Successful cultures can therefore be obtained only after their separation. Two conditions must be fulfilled to obtain unialgal or pure cultures: (1) the relevant or desired species must be isolated, and (2) it must be induced to multiply.

A. Biological Isolation

Biological isolation of algae can be attained through enrichment culture methods. Success in separating algae from other micro-organisms depends on whether, at some stage in the life cycle, a clean bacteria-free surface exists. In the Chlorococcales group, for instance, cells newly released from the parent cell wall are practically void of surface contamination. The same is generally true for most diatoms and flagellates.

Zoospores are usually considered the best material for pure culture. Zoospores of algae and motile states of flagellates can be expected: (1) when natural waters from running, well-aerated sources are taken to the laborato ; (2) when algal cells are transferred from moist to liquid media; (3) when fresh nutrients are added after exhaustion of nutrients in the old medium; and (4) when there is a change in light intensity.

B. Mechanical Isolation

Stein (1973) has described a number of isolation methods, the more popular of which include the capillary pipette method, streak-plating, and isolating on agar.

1. Capillary pipette method

This method uses an inverted Petri dish top as an isolation dish.

• place 10–15 drops of the natural collection in the centre of the dish

• place 6–8 drops of suitable liquid medium in six positions encircling the natural collection. Each droplet is then assigned a numerical code

• with the use of sterile capillary pipette, transfer the desired algal units from the natural collection to one of the six drops. Desired algal units are located while looking through an inverted microscope or a stereomicroscope

• transfer a single algal unit from the first drop to the second drop

• repeat the process (moving clockwise) until a single algal unit is present in a drop of liquid medium

• transfer the single algal unit to a sterile tube containing liquid culture medium.

2. Streak-plating

This method of isolation is recommended when size of desired algal species is about 10 micrometres or less.

• prepare Petri dishes containing sterile growth medium solidified by 1–1.5% agar

• place 1–2 drops of natural collection near the periphery of the agar. Flame-sterilize a wire loop or a glass rod using 70% ethanol and alcohol lamp. Make parallel streaks of the suspension of the agar.

• cover, incubate the plate 4–8 days under suitable conditions for growth

• through a stereomicroscope, observe and select the desired colonies for further isolation

• remove a sample of the desired colony using a wire loop and place in a drop of sterile medium on a cover glass. Observe under high power objective of a compound microscope

• repeat streaking procedure using samples from a single colony

• once growth is observed, transfer desired algal units to liquid or agar medium

3. Isolation on agar

Separation of desired algal units from contaminants can be attained by inoculating them on an agar (solid) surface or by allowing the units to isolate themselves by creeping away from the contaminants.

• prepare agar on Petri dishes

• remove the lids from the Petri dishes to allow the agar surface to dry

• place a drop of raw material near the centre of the agar surface

• while observing under a stereomicroscope, manipulate a single unit in a zigzag manner using a flame-sterilized fine needle until isolation is achieved

• draw the isolated unit using a capillary pipette or separate the unit by cutting a small agar block with a sterile microspatula

• transfer the isolated algal unit to a tube of liquid growth medium

PURIFICATION

Axenic cultures are usually required for biochemical and physiological studies of microalgae. Stein (1973) mentions a number of methods which will yield axenic cultures.

1. Centrifugation - repeated washings in a liquid medium, then the units are centrifuged.

2. Ultrasonic treatment - this method by Brown and Bischoff (1962) makes use of low intensity (90 K cycles/s) ultrasonic water both in which contaminants are physically separated from the units. This is followed by repeated washing and centrifugation.

3. Antibiotic treatment - this method is usually employed when purification by A and B fails.

   • prepare the antibiotic solution: dissolve 100 mg penicillin G (K or Na salt) and 50 mg streptomycin sulphate in 10 ml distilled water; add 10 mg chloramphenicol dissolved in 1 ml 95% ethanol to the penicillin streptomycin sulphate solution; mix well

   • filter the antibiotic solution using a membrane filter (millipore)

   • dispense 50 ml culture medium in six Erlenmeyer flasks (125-ml capacity)

   • inoculate 1 ml algal suspension

   • to each of the flasks add one of the following volumes of the antibiotic solution: 3.0, 2.0, 1.0, 0.5, 0.25, 0.125 ml. (This provides penicillin levels ranging from 20–500 mg/l and corresponding levels of the other two antibiotics used.)

   • place cultures in a “controlled room”

   • after 24 and 48 hours, aseptically transfer some algal units from each flask to tubes

   • place tubes under controlled laboratory conditions

   • check for bacterial contamination after 2–3 weeks

4. Treatment for other contaminants - unialgal cultures are often difficult to obtain due to the presence of other algae particularly those belonging to the Cyanophyceae and Bacillariophyceae groups. Lewin (1966) recommends the use of germanium dioxide (6 mg/l) which is added to the medium (germanium dioxide may be easily dissolved by using concentrated sodium hydroxide). Treatment should be discontinued as soon as diatom contamination is no longer observed.

The use of 70 ppm NaOCl (Purex brand) has also been proved effective in eliminating contaminants such as blue-green algae and protozoans. Streptomycin at 25 mg/l can also be used.

3.3 GROWTH

The growth as commonly applied to bacteria and other microorganisms usually refers to changes in the culture of cells rather than to changes in an individual organism. Growth denotes the increase in number beyond that present in the original inoculum (Pelczar et al., 1977). Four distinct phases of growth are described in Figure 1.

3.3.1 The lag phase: After the addition of inoculum to a culture medium the population remains temporarily unchanged. This does not imply that the cells are then quiescent or dormant. At this stage the cells increase in size beyond their normal dimensions. Physiologically, they are very active and are synthesizing new protoplasm. The organisms are actually metabolizing, but there is a lag in cell division. At the end of the lag phase, each organism divides.

3.3.2 The logarithmic or exponential phase: The cells here begin to divide steadily at constant rate. Given optimal conditions, the growth rate is maximal during this phase.

3.3.3 The stationary phase: Here the logarithmic phase of growth gradually begins to taper off after several hours (or days). The population remains more or less constant for a time, perhaps as a result of complete cessation of division or the compensation of the reproduction rate by an equivalent death rate.

3.3.4 The phase of decline or death: After the stationary phase, the rate at which cells die is faster than the rate of reproduction of new cells. Here the number of viable cells decreases geometrically.

The growth constant for a given species has a maximum value when measured under optimum growth conditions. It is decreased markedly by departures from the optimum conditions with respect to temperature, light, and the amount of micronutrients present.

Therefore, an important requirement of algal culture is the control of culture conditions. This need has brought about the use of controlled environment rooms where factors such as illumination, temperature and “day length” can be varied at will.

3.4 CONDITIONS AFFECTING GROWTH

3.4.1 Illumination

The production of organic matter in the sea by photosynthesis is dependent upon the intensity of incidental light at the sea surface and the depth to which adequate light can penetrate. For algae cultured in controlled rooms, cool-white daylight fluorescent lamps may be used. Two 8-ft, 40-Watt lamps will give a light intensity of about 300 foot candles (3 200 Lux) on a surface 16 inches away. For maintenance purposes, the light from northern exposure (northern hemisphere) may be used to light algal cultures.

Note: These north-facing windows must have no heating vents or radiators below them. If cultures are to be scaled-up for massproduction purposes, larger culture vessels may be subjected to ambient conditions of illumination (where dark and light regimes exist). Finally outdoor culture tanks will have to rely on sunlight for illumination.

3.4.2 Temperature

Temperature normally affects rate of metabolism of an organism. In controlled rooms, temperature is kept within the range of 18°–22°C. In outdoor cultures where temperatures are normally higher, the turnover rate is faster. Algal cultures when scaled-up may gradually be subjected to increases in temperature so as to avoid environmental stress. It is generally recommended that indoor to outdoor culture transfer be done early in the morning.

3.4.3 Culture Medium

Phytoplankton usually requires nutrients like nitrogen, phosphoius, potassium and other elements incorporated in the water medium in which it grows.

Stock or maintenance cultures make use of organic compounds in the form of thiamine (B1), Cynocobalamin (B12) and biotin.

Marine phytoplankton is grown in either enriched seawater media, or artificial or synthetic seawater media. Enriched seawater media make use of seawater as base plus small or trace amounts of elements essential for growth. Artificial media, on the other hand, have as a base distilled water plus known amounts of various elements to approximate seawater composition. It has been observed, however, that artificial media show the most constant result for algal culture in contrast to enriched natural seawater which may show varying results depending upon the time and place of collection.

Various media employed for the culture of phytoplankton and the steps involved in media preparation are described in Appendix 1.

3.4.4 Starter or Inoculum

Starter refers to the “seed” used to start algal cultures. The quality of the starter should be regularly checked for the presence or absence of contaminants. The amount of inoculum to be used is determined by the total volume of culture. In cases where culture conditions may be manipulated, small amounts of starter could be used. For large-scale algal production, however, more starter is required to effect faster harvest of cultures.

The growth period of a particular culture starts from the time the seed is introduced to the point in time where cultures are to be harvested or renewed. Renewal of cultures is necessary to ensure a continuous supply of phytoplankton for the hatcheries. A programming scheme for natural food culture is presented in Figure 2.

Fig. 1 The characteristic pattern of growth shown by unicellular algae in a culture of limital volume

Fig. 2 Programming of natural food culture for hatchery operations

Fig. 3 Schematic diagram of the initial culture method for rotifers, called “daily tanktransfer”. The method was developed in the Yashima Station of the Seto Inland Sea Farming Fisheries Association during 1964–1967. Several 0.5m³ slate tanks were used for producing both marine Chlorella and rotifers

Fig. 4 Conceptional diagram of mass-culture of rotifers in a “drain-off” system in large outdoor tanks

Appendix 1
CULTURE MEDIA AND THEIR PREPARATION

Note: - Dispense 2 ml each of solutions A, B, and C/litre of seawater
- Dispense 1 ml each of solutions D, E, and F/litre of seawater

Appendix 2
NUTRIENT MEDIA UTILIZED FOR VARIOUS PHYTOPLANKTON SPECIES IN SEAFDEC PHYCOLOGY LABORATORY

PREPARATION OF MEDIA

1. ENRICH SEAWATER MEDIA

   1. Add approximately 80–90% of the required volume of seawater to a beaker.

   2. Dispense the appropriate nutrients from previously prepared stock solutions.

   3. Check the pH and make necessary adjustments either 1N HCl or NaOH; ensure that the media are well mixed.

   4. Dilute the medium to volume with sterile seawater and dispense into containers.

2. ARTIFICIAL MEDIA

   1. Dissolve appropriate quantities of weighed salts in about 80% of the final volume of distilled water.

   2. Dispense other previously prepared solutions

   3. Add other nutrients.

   4. Check pH and dilute to volume with distilled water.

ZOOPLANKTON CULTURE

The more popular species of zooplankton utilized for fish seed production are Brachionus plicatilis and Tisbintra elongata

Brachionus plicatilis Muller

A. Taxonomic position
        Phylum Trochelminthes
            Class Rotifera (Notatoria)
                Order Monogononta
                    Suborder Ploima
                        Family Brachionidae
                            Subfamily Brachioninae
                                Genus Brachionus Pallas
                                    Species Brachionus plicatilis Muller

B. General Description of Rotifers

Most rotifers are microscopic aquatic animals. The body shape is extremely variable and is divided into three parts: head, trunk, and foot (post–anal portion). The entire integument is thin and flexible.

The head carries a corona, which is surrounded by cilia. The presence of cilia normally providesfor locomotory functions.

C. Potential

The rotifer Brachionus plicatilis is one of the most important zooplankton species presently utilized as live food for various cultivable marine animals.

D. Food and Feeding

Chlorella has probably been the most popularly used algal food for the culture of Brachionus plicatilis.

However, owing to difficulties frequently encountered in mass–producing sufficient supplies of phytoplankton species for rotifer culture, the possibility of utilizing other types of food was investigated. In 1967, Hirata and Mori introduced the use of baker's yeast for rotifer culture. In 1973, Hirayama and Watanabe also tested yeast, Rhodotorula sp., and a commercialized dried marine yeast (ASY–4011) concluding that yeast may be used most effectively as a supplemental food when phytoplankton supply is not sufficient. Another marine yeast species, Zygosaccharomyces marinus, isolated by Kawano (1968), was also found to be a good food for Brachionus (Furukawa and Hidaka, 1973; Hirata, 1974) and zoeal larvae of P. japonicus (Furukawa, 1972).

E. Culture Techniques

1. The “daily” tank–transfer method

In this method Brachionus is continuously subcultured using 0.5 m tanks. The tanks are initially used for Chlorella cultures. When Chlorella density reaches about 10–20 × 10 cells/ml inoculation of rotifers are harvested and transferred to another tank (see Figure 3). The process of transfer of subculturing continues for an indefinite period. The main disadvantage of this method is that it is too labour–intensive.

2. The “drain–off” system for large tank outdoor culture

1. culture marine Chlorella using modified Hirata medium until density reaches 10 x 20 cells/ml

2. fertilize again using double strength of abovementioned culture medium

3. when Chlorella reaches stationary phase of growth, Brachionus is inoculated at an initial density of 10–50 individuals/ml

4. once Chlorella is consumed, introduce additional food or baker's yeast (at 1 g/million rotifers/day)

5. when Brachionus density reaches 120–150 ind/ml_x, harvest about 20–30% of the culture and transfer to another tank

The procedure is thus repeated until the water becomes polluted with materials. A diagram of the “drain–off” system is shown in Figure 4.

3. The feedback culture system

Hirata (1979) describes feedback as “feeding resulting in excretion, then excretion converted into feeding again”. In an artificial culture system, “self–purification” which is carried out smoothly in the natural waters, cannot be attained due to the accumulation of biodeposits produced by the cultured organisms. Therefore, a system should be devised to promote energy flow and this is the principle from which Hirata's feedback system evolved. A flow chart of the principle of the feedback culture system is presented in Figure 5.

In a feedback system, marine zooplankton such as Brachionus and Tigriopus japonicus (or the harpactocoid copepod Tisbintra elongata) may be cultured together. These organisms serve as consumers. The microalgae Chlorella and the macroalgae Enteromorpha intestinalis are initially cultured as producers. Bacteria which grow on the tank will then serve as decomposers.

There are two tanks in this system - a circular tank which serves as reservoir and a rectangular tank called the zigzag stream unit. The water in the first tank is recirculated to the stream by an airlift pump at the rate of 20 times a day. The macroalagae Enteromorpha intestinalis are grown together with the zooplankton in the zigzag stream. Frame nylon nettings are placed in each of the slots in the stream to serve as attachments for Enteromorpha which is filamentous. Water quality can be maintained for long periods of time in this type of culture system.

Both the drain–off and the feedback culture systems are employed for the mass culture of Brachionus plicatilis and copepod Tisbintra elongata.

For outdoor culture of Brachionus, 1-t fibreglass tanks are used and rotifers rely mainly on Chlorella virginica for food. The use of baker's yeast is also employed especially when supply of Chlorella is insufficient. The use of other types of food is presently being experimented.

Tanks are regularly cleaned and disinfected using diluted (about 10 percent) muriatic acid solution. Thorough rinsing is done prior to use. During rainy months the tanks are usually provided with framed plastic sheets which serve as covers.

Water utilized for culture purposes undergoes filtration provided by improvised sand filters.

Water salinity for rotifer culture is usually within the range of 25–26 ppt.

Water temperature ranges between 27° and 30°C.

Table 1

HIRATA MEDIUM

^* Clewat 32 is the commercial name given to a formulation consisting of Fe, Zn, Mn, Co, Cu and Bo with EDTA

Table 2

NUTRIENT MEDIUM FOR CHLORELLA VIRGINICA OR TETRASELMIS SP.