Introduction

The early life stages of seabass and gilthead seabream are zooplankton-feeders, i.e. they prey on small free living planktonic animals. As no artificial larval diet can at present totally fulfil their nutritional requirements, their successful rearing still depends on an adequate supply of high quality live feeds, usually in the form of rotifers (fed on unicellular algae) and brine shrimp (Artemia spp).

This chapter describes the equipment and operation to mass produce these organisms, whose biology has been presented in Part 2. The design of the hatchery sections for production of live feed is described in the Design and Engineering part of this manual.

The technology for phytoplankton and zooplankton mass production has become very reliable and the production of live feeds is part of the standard working procedures in Mediterranean hatcheries. The efficiency of this part of the hatchery mainly depends on the implementation of standard procedures by well trained staff.

As live preys for first postlarval stages of seabass and gilthead seabream, two small animals are extensively used:

• all-female (amictic) populations of the rotifer Brachionus plicatilis (60 - 350 mm in length);
• larval stages (nauplii and metanauplii) of a small crustacean, the brine shrimp Artemia spp. (length: 400 - 800mm).

In farms dealing with seabass only, live feed production is often limited to the hatching of Artemia nauplii, which are obtained by incubating their dry resting eggs (cysts). The mouth of the seabass at first feeding is large enough to gulp brine shrimp nauplii and these larvae do not require the smaller rotifers as first feed, as it is in the case of gilthead seabream. The hatcheries working on both species, or on gilthead seabream alone, have to produce rotifers as well as microalgae.

As said in previous sections of the manual microalgae are now used not only for rotifer production (see below), but also to improve water quality in the larval tanks, creating the so called “green water”, which is used during the initial rearing phases.

In case of rotifer mass production, clear advantages of this organism are given by its fast reproductive rate and by the high densities that can be reached in the rearing facilities, up to 1,000 rotifers per ml and over. The daily increase of their populations ranges between 50 and 150%, depending on the production technique chosen and the nutritional value of their diet. Their main drawback is that to culture them microalgae are needed as food, at least in the initial steps. However, for their final production process in large volumes (see below) there are now good artificial feeds which can replace algae, whose mass production remains unavoidable at least in gilthead seabream production for the “greenwater.

Fig. 23.00 A sort of simplified trophic chain is established in the hatchery.

On the other hand, the production of Artemia is greatly facilitated by the availability of dry resting eggs, which can be purchased from specialised suppliers. If properly canned and stored, brine shrimp cysts can remain viable for years.

As rotifers and nauplii are produced to fulfil the needs of the larval rearing unit, they have to be available at given times, in pre-set quantities and with their nutritional quality intact. To achieve this, the design and operation of the culture systems should pay special attention to the following points:

• adequate dimensioning of the production facilities, including additional space for back-up cultures as a precaution against culture collapses;
• proper daily renewal and up-scaling of the cultures (standard operating procedures);
• maintenance of strict hygienic conditions in the culture environment (cleaning procedures);
• close control of culture conditions (monitoring procedures).

Mass culture of microalgae

Mass production of phytoplankton for rotifers and “green water” in most Mediterranean hatcheries is limited to a few species such as: Chlorella sp, Isochrysis galbana, Pavlova lutheri, Nannochloropsis oculata and N. gaditana, Dunaliella tertiolecta and Tetraselmis suecica. These species have been selected on the basis of their size, nutritional value, culture easiness and absence of negative side effects, such as toxicity. Their nutritional value shows a great variability not only among different species, but also in genetically different populations of the same species (strains). For hatchery purposes, the species to be cultured should both fit well the local rearing conditions and have a high nutritional value for rotifers. The increasing availability of nutritional boosters as enrichment diets for both rotifers and brine shrimps, has made this choice easier.

Fig. 23.01 Mass culture of microalge (photo STM Aquatrade)

Population dynamics

Microalgae population dynamics can be described by different phases:

•  the lag-phase, where, just after the inoculum, the cells increase in size, but not in number, and begin to absorb the nutriens supplied with the culture medium;
• the log-phase (or esponential phase), where cells reproduce very fast and population growth is exponential;
• the transitional phase (or declining growth phase), where growth rate slows down;
• the stationary phase, where cells remains constant in number and reproduction is balanced by death;
• the decline phase, where cell number decreases since death rate exceeds growth.

It is advisable to harvest phytoplanktonic organisms during their log phase, since in the new culture they will grow more rapidly and will yield a more viable population.

Mass production systems

For aquaculture purposes, microalgae are mass produced in three main ways: (i) batch (or discontinuous or multistep back-up system) culture, (ii) semi-continuous culture, and (iii) continuous culture.

In the batch culture a small axenic stock culture produces a series of cultures of increasing volume where the algal population of each culture vessel is entirely harvested at or near its peak density, i.e. while still conserving a good growth potential, to be used either as inoculum for other culture vessels, or to feed rotifers or be used in fish larval tanks. It typically makes use of small (few liters) to medium size (500 liters) containers, and it is kept indoor and under strictly controlled, if not properly axenic, conditions. It is considered by many authors the easiest and most reliable method of algal production, provided that the working protocol is strictly enforced. Algal quality is less erratic than in the semi-continuous method, even if the latter is more productive for any given volume.

In the semi-continuous system the algal population, when mature, is partially harvested at intervals. The harvested culture volume is replaced by fresh medium to keep growth going on. This culture is adopted to produce large amounts of algae and frequently uses large outdoor tanks. Their main drawbacks are: (i) the unpredictable duration, (ii) the risk of contamination by other organisms as competitors (other microalgal species), contaminants (bacteria) and predators (ciliate protozoa feeding on the algae), as well as (iii) the building up of metabolites, which can affect quality.

The continuous system is a steady-state continuous flow culture in which the rate of growth is governed by the rate of supply of the limiting factor. It is a balanced axenic system where the algal population is harvested and fertilised continuously. This method, though the most efficient over extended periods, produces limited amounts of high quality cells and requires complex equipment as well as advanced management. A relatively recent development of this system is represented by the photo-bioreactor, a continuous culture device that increases the density of cultured microalgae to very high levels under predictable environmental and microbiological conditions.

The microalgae produced can be concentrated to a dense liquid suspension by centrifugation, and can then be stored for more than one month in the refrigerator, still giving excellent viability when used. A new industry is now appearing, whose concentrated algal products can also fulfil the hatchery needs, saving the time-consuming and expensive production of microalgae in the hatchery.

The system described below is the batch culture, by far the most widely adopted method by Mediterranean hatcheries. Before its description, additional instructions are given concerning facilities, the preparation of the culture medium, and the equipment required.

Fig. 23.02 Old fashioned unit using artificial light for algae mass culture (photo M. Caggiano)

Mass culture facilities for microalgae

Algae are cultured in a dedicated sector of the live feeds production section, which is made of three working areas inside the hatchery building: a lab for duplicating small cultures, a conditioned room to maintain small culture vessels and pure strains and finally a large area for the mass cultures in PE bags or, less frequently, tanks. In the warmest Mediterranean areas, a light greenhouse can replace the latter.

Small volume cultures are kept in vessels ranging from 20-ml test tubes up to 18 l carboys. They can be made of borosilicate glass, polycarbonate, PET or any other material able to stand a sterilization process. These vessels are placed on glass shelves lightened by fluorescent tubes and equipped with a CO2 enriched air distribution system.

Hot-extruded tubular PE film is utilised for larger volumes bags. The film is usually 0.25 mm thick and its stretched width ranges from 45 to 95 cm. Two bag designs are widely adopted in Mediterranean hatcheries: the smaller suspended bag and the larger one placed within a steel wire cylindrical frame. The first type has a capacity of 60 I (single) to 150 I (double or U-shaped), whereas the latter, that stands on a saddle-like GRP base to improve circulation, can contain up to 450l. Their top is closed by a plastic cover to prevent contamination.

Fig 24.00 A typical scheme of a batch type production

All units are equipped with artificial lights, usually fluorescent tubes, an aeration system, often with an additional source of carbon dioxide, and stands for the culture vessels, i.e. light shelves for small volumes and metal racks or wired frames for PE bags.

The unit also stores the special equipment to process pre-treated seawater, such as fine filters and sterilizers, as well as a laboratory where nutrients and glassware are prepared and stored, and where the necessary monitoring operations are performed. Standard cleaning procedures have to be strictly followed to maintain proper hygienic conditions (see Annex 6).

Preparation of the culture medium

In planktonic mass production, seawater is the medium of all culture vessels. The use of other mediums such as agar is limited to the preservation of pure algal strains. Ideally, seawater should be free from pathogens and pollutants. With this aim in mind, seawater is treated to remove suspended solids, contaminants and organisms and to improve its original parameters to fit the quality standards set for proper growth of microalgae. These methods are outlined below, while the description of related technical equipment can be found in the Engineering part of the manual.

Mechanical filtration

The most common systems of seawater mechanical treatment (applicable to all culture volumes: strains, small volumes and mass cultures) for microalgae production (which also apply to rotifers and brine shrimps cultures), are described below.

Raw seawater is first pre-treated to remove the bulk of suspended solids and contaminant organisms. Different methods are followed, but the most commonly used is a combination of settling and sand filtration. If properly dimensioned, a settling tank is a useful device. Not only it improves the water quality at no cost by decanting its suspended matter, but also provides a reservoir of seawater to tackle unpredictable problems in seawater supply, such as a damaged main pump station or a temporary contamination of seawater (oil spill, river plume, exceptional storm). Settled water is then filtrated through a sand or bag filter that retains particles as small as 50,10 or 5 mm depending from the sector of destination. At this point filtered water can be used for most purposes in the hatchery, provided for some sectors like pytozooplankton it undergoes UV sterilisation before use. In the live feeds sector a higher degree of filtration is required and pre-treated water is further micro-filtered to a size of 5 mm for large culture volumes and down to 1 mm for small volumes and strain cultures For this finer filtering polyethylene wire cartridges, bag filters or diatomaceous earth filters are used. Such a fine filtration should even remove bacteria and other micro-organisms, but in reality the filtering capacity is not absolute and cannot totally guarantee such results, in particular under hatchery working conditions. It is therefore recommended to proceed with the final step, sterilization of filtered seawater.

Fig. 24.01 Mechanical sand filter for the water inlet (photo STM Aquatrade)

Sterilization

Different methods of water sterilization have been developed. The following description refers to the most common methods adopted for hatcheries. The choice is based on local availability of equipment and service and depends also on the amounts of water to sterilize, which are related to the size of the hatchery.

UV light sterilisation (applicable to all culture volumes)

UV light with a wave length of 265 nm (short wave UV or UV-C) has a strong germicidal effect based on its capacity to break the DNA helix. It is produced by special high or low pressure mercury vapour lamps whose germicidal capacity depends on several factors such as their power, seawater transmittance (transparency to UV), type and quantity of microorganisms to be destroyed, degree of purification required, water flow (contact time) and temperature. Seawater to be sterilized flows through one or more sealed chambers where it is irradiated by one or more lamps placed inside quartz tubes (transparent to UV light). The thickness of the water film inside the irradiation chamber should be such as to allow the maximum sterilization effect. If the power of UV lamps and chamber design are properly dimensioned, the contact time between water and زquartz tubes is only of few seconds.

Fig. 24.02 Compact water treatment unit for zooplancton (photo STM Aquatrade)

For practical purposes, and under normal hatchery conditions, an intensity of at least 40 mJ/cm2, provided at the end of the life span of the lamps, removes 99% of most unwanted micro-organisms for fish farming from the treated water volume. With its auxiliary equipment (manual or automatic wipers, UV sensors and stabilizers, computer-aided control), this method is very effective and manageable and fully justifies its cost.

Chlorine sterilization (applicable to all culture volumes)

Active chlorine is a strong oxidizing agent, commercially available as liquid bleach (sodium hypochlorite or NaOCl) and as bleaching powder (CaOCl2). The percentage of active chlorine in these chemicals should always be checked in advance as it changes widely according to the producer: commercial grade NaOCl usually contains 5-15% active chlorine, while CaOCl2 contains 60-70%. Annex 7 describes how to prepare hypochlorite solutions, to assess their active chlorine content and the residual chlorine in treated water, as well as the methods to sterilize seawater and deactivate residual chlorine.

Independently from the method employed, a final dosage of 5 to 10 ppm of active chlorine is used to sterilize seawater. The contact time between water and chlorine should be at least one hour, after which any residual chlorine must be neutralised with sodium thiosulphate, Na2S2O3, (see Annex 7 for details). This technique is now widely used as final sterilisation step of water in larger vessel and of the culture equipment (air and oxygen tubing and زdiffusers, detritus traps, submersible water heaters)

Fig. 24.03 Chlorine container for laboratory use (photo STM Aquatrade)

Use of an autoclave, or wet vapour sterilization (applicable to small volume cultures)

With this method, applicable only up to 5-6 I volumes depending on the autoclave size, seawater is sterilised together with the culture vessels, usually made of Pyrex® glass, due to their resistance to heat. The autoclave should work at 120°C under a 2 atm pressure. Sterilization time ranges from 10 min (100-ml flasks) to 20 min (200-ml flasks) and 30 min (up to 5-6 I vessels). The neck of each container has to be covered with a loosened aluminium foil stopper to let vapour out during the sterilization.

Dry vapour sterilization (applicable to small volume cultures)

This method replicates the previous one, but the autoclave is replaced by an oven. As it works in dry vapour at ambient pressure, glass vessels filled with seawater are heated at 160-170°C for 2 to 3 hours. Dimensions and stoppers of vessels to be sterilized are the same ones used for wet vapour sterilization.

Enrichment

The exponential growth of the microalgal populations is regulated by four most important parameters: light, pH, turbulence and nutrients. Whereas the first three can be easily adjusted specific nutrients have to be added to the culture medium in proper quantities.

The main nutrients required are nitrogen (N) and phosphorus (P), followed by trace minerals, vitamins and chelating agents. Nutrient solutions are prepared in advance according the type of microalgae cultivated. With the exception of N and P solution, the other nutrients are stored as primary stock solutions, which are used to prepare the working solutions according to the day-by-day production schedule. Aseptic conditions have to be maintained in the preparation of the enrichment solutions. The vitamin solution cannot be sterilised because heating will deactivate the vitamins. The microalgae selected for the reproduction of seabass and gilthead seabream require the following fertilizers that refer to the enrichment medium Guillard f/2.

Primary stock solutions

Trace elements and vitamins are first prepared as concentrated primary stock solutions: in this way, if properly stored, they may last several months. Trace elements are prepared as four different solutions, each stored, like vitamins, in a separate container. To prepare one litre of each stock solution, the following quantities (in grams) are required.

Trace element stock solutions

Solution A: ZnSO4 · H2O (30g) + CuSO4 · 5H2O (25g) + CoSO4 · 7H2O (30g) + MnSO4 · H2O (20g)

Solution B: FeCl3 · 6H2O (50g)

Solution C: Na2MoO4 · 2H2O (25g)

Solution D: EDTA · 2H2O (50g)

To prepare the solution put the components, according to the proportions indicated above, into one 1 -I graduated Pyrex® bottle and fill with distilled water (DW) to the mark. Deionized water can also be employed if distilled water is not available. When the components are fully dissolved, store at ambient temperature, avoiding exposure to direct light.

Vitamins stock solutions

B12 Cyanocobalamin (0.1 g)

B1 Thiamin (10g)

H Biotin (0.1g)

Place the indicated quantity of each vitamin into a sterilized 1 -I graduated Pyrex® bottle filled to the required volume with sterilized DW. When fully dissolved, store in refrigerator and keep away from light. The B12 solution should preferably be stored in a dark or aluminium wrapped bottle.

Warning: do not sterilize any vitamin solution.

Working solutions

The working solutions represent the way to add the nutrients, trace elements and vitamins directly to the seawater medium. Two working solutions are prepared by diluting the above mentioned stock solutions, whereas the third solution is prepared directly from industrial grade chemical salts of N, P .and K.

Fig. 24.04 Working solutions ready to use (photo STM Aquatrade)

Mineral salts working solution

Mineral salts solution: NaNO3 (300g) + KH2PO4 (30g) +NH4Cl (20g)

Put the salts into one 1-I screw-capped oven-resistant glass bottle and fill with DW to the mark. If not available, deionized water can also be employed. When fully dissolved, sterilize either in autoclave or oven. Store at ambient temperature, avoiding direct light. Due to the comparatively higher requirements for the mineral salts stocking solution, quantities in excess of one liter are usually prepared at one time. Use 5 to 10-l glass autoclavable vessels, then store in 5- or 10-I plastic carboys with bottom tap.

Use: 1 ml per litre of seawater medium.

Trace elements working solution

One liter of trace element working solution is prepared according to the sequence and quantities oulined below:

Solution D (100 ml) + Solution A (10 ml) + Solution B (10 ml) + Solution C (10 ml).

Use only sterile glassware: a 100-ml cylinder and three 10-ml pipettes (one per solution). Mix the four solutions into a 1-I screw-capped oven-resistant glass bottle and fill with DW or deionized water and sterilise either in autoclave or oven. Store at ambient temperature, avoiding direct light.

Use: 1 ml per litre of seawater medium.

Vitamins working solution

Mix the following amounts of vitamins stock solutions: solution B12 (10 ml) + solution B1 (10 ml) + solution H (10 ml).

Use only sterile pipettes, one per vitamin. Dilute the mix to one litre volume with sterilized distilled water. Pour in a dark sterilized bottle (or wrapped in aluminium foil) and store in the refrigerator just before use.

Use: 1 ml per litre of seawater medium.

Warning: do not sterilize any vitamin solution

Culture equipment sterilization

As for water medium and nutrients, also the equipment should be kept clean and disinfected to prevent contamination.

Small glassware such as volumetric pipettes, Petri dishes, Pasteur pipettes and beakers, are sterilized either in an autoclave or in an oven. Bigger glass containers as Erlenmayer flask, balloons and carboys are sterilized after being refilled with seawater. Pipettes are divided by volume capacity and stored into capped metal containers or wrapped in aluminium foil. Polyethylene (PE) bags are considered sterile as they are obtained by hot extrusion and do not need special treatments. Tanks, plastic jugs for nutrients and piping for pump .transfer are disinfected with hypochlorite.

Fig. 24.05 Pipette container used with oven sterilization (photo STM Aquatrade)

All up-scaling tools and consumables that may be in contact with algae (aluminium foil, platinum needles, necks of tubes, flasks and balloons) are sterilized by flame using a Bunsen burner. On the glassware neck, flaming should lasts until all water drops have evaporated.

Sterilized hydrophobic cotton is commonly used as disposable stoppers for all glass containers. For its sterilization, it is packed in aluminium foil and sterilized in autoclave or oven. Cotton stoppers are disposed of after use. Hygiene and cleaning procedures in the live feed production sector are outlined in Annex 6.

Enrichment of culture vessels

This section describes how to proceed with the fertilization of the different culture vessels. All working nutrient solutions are diluted at 1 ml per litre of seawater medium. Pure strain cultures in test tubes, in marine agar and their initial small volumes are fertilized with half dosage (0.5 ml/l)..

Fig. 24.06 Phytoplankton strain cultures (photo STM Aquatrade)

With small vessels, this operation should be carried out in a dedicated room kept clean and equipped with all the necessary tools, glassware and consumables. To reduce the risk of contamination, a UV-light ceiling lamp can be installed to provide germicidal irradiation when the room is not in use.

Enrichment procedure for small vessels (up to 20l capacity):

1 prepare on the bench the required vessels containing sterilized water, nutrient working solution and the necessary tools;

2 carefully loosen their aluminium foil caps;

3 heat each flask neck and the inner side of the aluminium foil caps with a burner; close again;

4 heat opening and screw cap of each bottle containing stock solution in the same way;

5 take a sterile pipette from its sealed container (if in a metal case, open and close it at the flame);

6 with the pipette place one nutrient solution at a time in each flask, using a new pipette for each solution;

7 repeat the flaming of necks and aluminium caps on the enriched flasks.

Enrichment procedure for polyethylene bags (up to 500l capacity):

1 The bags are filled with the same treated and UV-light sterilized seawater utilised for small V vessels, the only difference being that the bags are not sterilized. Due to the larger amount of nutrients involved, use clean and sterilized graduated cylinders to fertilize bags.

2 Cut a 20 cm-long slit on the upper rim (empty part) of the bag;

3 add 1 ml per liter of each solution.

When cutting the bag, bear in mind the final culture level, i.e. after the addition of the inoculum.

Batch culture of microalgae

Mass culture of phytoplankton starts from pure strains of selected species and proceeds by upscaling from small (0.5 I) up to large volumes in PE bags (450I) or tanks (1 000 l and up). In finfish breeding, where large amounts of microalgae are not required, the last upscaling step is the PE bags. See annex 8 for a typical example of microalgae upscaling protocol, and annex 9 for the daily .workplan and culture file for microalgae.

Fig. 24.07-8 Flask sterilization and enrichment (photo STM Aquatrade)

Pure strain culture

The culture of pure strains of the selected microalgal species is the starting point of the mass production process. Strain quality is therefore essential for any successful production process. A good selection of pure strains of different algae should always be kept in a dedicated facility in the hatchery. This practice also allows the selection of the most suitable strains under local conditions and at a given time.

Pure guaranteed strains of algae, as well as of rotifers, are normally available from a few laboratories and institutions in the Mediterranean region and Europe. It is strongly recommended to regularly renew their lines, not only in case of culture crashes, but also to control the often unavoidable contamination or decline in quality. Strains from other hatcheries should better be avoided because of their possible decline in quality and the associated risk of introducing contaminated strains.

The algal pure strains are kept under standard controlled environment in conditioned rooms or in especially designed incubators, in which routine work can be performed under strict hygienic control. The pure strain cultures are usually kept in small glass containers, such as 10 to 25 ml test tubes or 100-ml glass Erlenmeyer flasks, closed by a sterile stopper (screw cap or a folded aluminium foil).

Pure-strains cultures should be maintained at a steady or resting stage, i.e. under environmental conditions which allow them to reproduce, but not to increase exponentially in number. In this way, their sexual reproduction is fostered, thus increasing their genetic variability, and the growth of unwanted organisms such as other algal species, bacteria and ciliate protozoa is prevented. Culture parameters are therefore kept below the values adopted for mass production. In particular, only half dose of nutrients is used, water temperature is kept at around 14-16°C, light intensity ranges from 300 (test tubes) to 1 000 lux (flasks) and no aeration and carbon dioxide are provided.

Under routine conditions, strain cultures are usually renewed every month. In the replication process, an inoculum of 0.1-0.2 ml (from test tubes) or 0.5 to 1 ml (from Erlenmeyer flasks) is taken from the best old culture which is free of contamination, to inoculate three new vessels of the same size to start a new strain. The old culture is then either utilised for upscaling, or is discarded. Strain culture vessels should be stirred at least once a day by hand, paying attention not to stir bottom debris up.

Warning: in the management of pure algal strains quality is essential: get rid of any tube or flask which is found to be contaminated by bacteria, fungi, ciliates, nematodes or different algal species.

Amany Esmail

Manger GAFRD Web Site