Part 3Hatchery operation: culture of algae
Unicellular marine microalgae (Figure 12) are grown as food for the various stages in the hatchery culture of commercially valuable shellfish. Until recently living algae constituted the sole food source for bivalve larvae and juveniles. This is now beginning to change as the result of recent research into the development of suitable non-living and artificial diets. However, the production of live algae will remain a critically important aspect of successful hatchery management into the foreseeable future, if only as a live food supplement to innovative foodstuffs.
Figure 12: Photomicrographs of two algal species commonly cultured in hatcheries, Isochrysis sp. (A) and Tetraselmis sp. (B) showing the relative difference in cell size.
Flagellate and diatom species, among the microalgae, are primary producers at the base of the marine food chain. They manufacture organic cellular components from the uptake of carbon dioxide and nutrients contained in seawater using light as the energy source in a process called photosynthesis. They are normally cultured in hatcheries in suitably treated natural seawater enriched with additional nutrients, which include nitrates, phosphates, essential trace elements, vitamins and carbon dioxide as the carbon source. Synthetic seawater may be used but it is prohibitively expensive except at the small laboratory scale.
The need to culture microalgae arises because the natural phytoplankton content of seawater used in the hatchery is insufficient to support optimum growth of high densities of larvae and juveniles reared. Particularly in the culture of larvae, the water treatments used will remove almost all of the natural phytoplankton which then needs to be replaced from cultures of preferred, high food value species. In this context, and in the provision of suitable food rations for breeding stock and juveniles, few of the very many naturally occurring algae are of good food value to bivalves and not all of these are amenable to artificial culture on a sufficiently large scale. A list of the more commonly used species in bivalve hatcheries is given in Table 1. Parameters of cell size and composition are also shown.
Table 1: The cell volume, organic weight and gross lipid content of some of the more commonly cultured algal species used as foods for bivalve larvae and spat. Species marked * are of relatively poor nutritional value.
The culture of algae accounts for about 40% of the costs of rearing bivalve seed to a shell length of about 5 mm in a hatchery. For example, 1 million juvenile Manila clams or Pacific oysters of 5 mm shell length will consume 1 400 l of high density, cultured algae each day at the optimum rearing temperature of 24°C. Smaller daily volumes are required to feed broodstock and larvae.
The basic methods of algal culture have changed little over the years and the various steps in the process leading to production-scale cultures are introduced in Figure 13. Hatcheries have either opted for indoor, intensive culture with artificial illumination, usually external to the culture vessels, or outdoor, extensive culture in large tanks or ponds utilizing natural light. The intensive techniques are satisfactory in terms of reliability and productivity but are expensive in terms of capital outlay and labour, while the extensive methods tend to be less reliable and, sometimes not very productive. Both methods will be considered together with the essential infrastructure and methodologies. A schematic diagram of the process of culturing algae is given in Figure 14 and a floor plan of a hatchery showing the area allocated to algal culture was given earlier in Figure 5 (section 1.2).
Figure 13: Steps in the production of algae. Stock cultures (250 ml or less) remain in isolation under light and climate control (low temperature) and are only used to inoculate starter cultures when necessary. They are not aerated nor is carbon dioxide added. Starter cultures (250 ml to 4 l in volume) are grown quickly for 7 to 14 days at higher temperatures and light intensity with a supply of carbon dioxide enriched air. When ready, a small portion of the volume is used to start a new starter culture and the main portion to begin an intermediate-scale culture. Intermediatescale cultures (usually of between 4 l and 20 l in volume) may be used as food for larvae or to start a large-scale culture. Large-scale cultures are generally of a minimum of 50 l and are frequently much greater in volume.
Figure 14: The process of algal culture showing the various required inputs. Whether secondary seawater treatment is necessary or not depends on the extent to which the water is initially filtered.
3.2 MAINTENANCE OF STOCK AND STARTER CULTURES
Stock cultures, otherwise known as master cultures, of the preferred species are the basic foundation of culture. They are normally supplied as monospecific (uni-algal) cultures from reputable culture collections maintained by national institutions or research laboratories. Since they are valuable, they are normally kept in specialized maintenance media, for example, Erdschreiber, or alternatively in F/2 media, or on nutrient enriched agar plates or slopes, under closely controlled conditions of temperature and illumination. A special area or room off the algal culture room is usually allocated to this purpose.
Stock cultures are used only to provide lines of starter cultures (also known as inocula) when required. Every effort should be made to minimize the risk of contaminating the stock and starter cultures with competing microorganisms. The sterile procedures described below should be followed to ensure that contamination does not occur.
Stock cultures are kept in small, transparent, autoclavable containers. For example, 500 ml borosilicate glass, flat-bottomed boiling or conical flasks fitted with a cotton wool plug at the neck, suitable for containing 250 ml of sterile, autoclaved medium, are ideal. The composition and preparation of Erdschreiber medium is given in Table 2. Alternate media suitable for the purpose are Guillard’s F/2 (see Table 3) and HESAW (see Table 4). Proprietary algal culture enrichment products for addition to suitably treated seawater can also be used according to the manufacturer’s instructions. Stock cultures are also often maintained in seawater agar medium impregnated with suitable nutrients in Petri dishes or on slopes in test tubes.
Figure 15: Illuminated, temperature controlled incubators for the maintenance of small algal cultures.
Stock cultures are best kept in a cooled incubator at 4 to 12°C (according to preference), illuminated by two or more 8-watt (W) fluorescent lamps that provide a light intensity of 450 lux measured at the culture surface (Figure 15). Alternatively they can be kept in cool conditions close to a north-facing window (out of direct sunlight), or in a cool room illuminated by fluorescent lamps. The objective is not to allow rapid growth, but to maintain the cultures in good condition. The cultures are not aerated, nor is carbon dioxide introduced.
3.2.1 Procedures for the management of stock cultures
It is necessary to sub-culture stock cultures at monthly intervals to maintain them in a vigorous and healthy state. Following removal of the cotton wool plug from a stock culture flask and flaming the neck of the flask with a Bunsen burner (or butane torch), an inoculum of 20 to 50 ml is decanted into another sterile flask containing autoclaved medium. The plug is inserted after flaming of the neck of this new flask. Species name and the date are indelibly marked on the flask, which is then returned to the incubator. The original stock culture can be kept for a few weeks in the event that the new stock culture fails to grow. The stock culture transfer procedure is best performed in a cabinet that has been sterilized by ultra-violet light to further reduce the risk of contamination (see Figure 16). Details of the transfer procedure are given in the accompanying box.
Figure 16: A - schematic diagram of a culture transfer chamber. B - an autoclave suitable for the sterilization of small volumes of culture medium.
Table 2: The composition and preparation of Erdschreiber culture maintenance medium.
1. Seawater: Autoclave 2 l in a 3 l borosilicate glass flat-bottomed boiling flask with cotton wool plug at 1.06 kg cm-2 for 20 minutes. Stand for 2 days.
2. Soil extract: prepared as follows:
a) mix 1 kg soil from a woodland or pasture area untreated with artificial fertilizers, insecticides, etc. with 1 l of distilled freshwater;b) autoclave at 1.06 kg cm-2 for 60 minutes;c) decant off the supernatant liquid;d) filter supernatant through Whatman No. 1 paper and then through a glass-fibre (GF/C) paper;e) autoclave in 1 l aliquots in polypropylene bottles at 1.06 kg cm-2 for 20 minutes;f) store in deep freeze until required;g) autoclave 100 ml in 500 ml borosilicate glass, flat-bottomed boiling flask with cotton wool plug at 1.06 kg cm-2 for 20 minutes.
3. Nitrate/phosphate stock solution: Dissolve 40g NaNO3 and 4 g Na2HPO4 in 200 ml distilled water. Autoclave in 500 ml flask at 1.06 kg cm-2 for 20 minutes.
4. Silicate stock solution: Dissolve 8 g Na2SiO3.5H2O in 200 ml distilled water. Autoclave in 500 ml flask at 1.06 kg cm-2 for 20 minutes.
Add 100 ml soil extract (2) to 2 l of sterilized seawater (1). With sterile pipette add 2 ml nitrate/ phosphate stock solution (3) and 2 ml silicate stock (4). Decant 250 ml into 8 empty autoclaved 500 ml flasks with cotton wool plugs. Use a Bunsen burner or butane torch to flame the necks of the flasks immediately before and after decanting/pipetting. The maintenance medium is now ready to use.
Procedure for transferring algal cultures from flask to flask
(a) Wipe all inner surfaces of inoculating booth with 85% ethanol.
(b) Place all flasks that will be required in the booth; i.e. all flasks to be transferred from (the transfer flask) and flasks containing sterilized media to be transferred into (new flasks).
(c) Close booth and switch on ultra-violet lamp. Leave for at least 20 minutes. (It is not safe to look directly at ultraviolet light, so a dark cover should be placed over the plexi-glass (transparent acrylic plastic) viewing plate when the light is on.)
(d) Switch off lamp. Ignite small burner.
(e) Remove foil caps from one transfer and one new flask. Flame the neck of each flask by slowly rotating the neck through the flame.
(f) Tilt the neck of the transfer flask toward the new flask. In one motion, remove both stoppers and pour an inoculum into the new flask. Transfer approximately 50 ml for diatom species and 100 ml for flagellates. Avoid touching the necks of the two flasks. Never touch the portion of the stopper that is inserted into the flask. Once the inoculum is added, replace the stopper in the transfer flask. Slowly flame the neck of the new flask before replacing its stopper.
(g) Replace foil cap over the neck of the new flask. Using a waterproof marker pen, label the new flask with the algal species inoculated and the date of transfer.
(h) Repeat procedure for all flasks within the booth. Once completed, turn off burner and open booth.
(i) Remove all new flasks and place in the algal incubator or a well-lit area in the algae culture facility.
(j) The remaining inoculum in the transfer flasks can be used to inoculate larger cultures such as 4 l flasks or carboys.
(from: Bourne, Hodgson and Whyte, 1989)
3.2.2 Starter culture manageme
Figure 17: Photographs showing typical facilities for maintenance of starter cultures.
Procedures for the maintenance of starter cultures (inocula) are almost identical to those described above. These cultures are specifically grown to provide inocula to start larger volume cultures needed to produce food.
A line of starter cultures is originally set-up from the stock culture of the required species. Starter cultures, like the stocks, can be grown in 500 ml boiling flasks in 250 ml of culture medium. Because they are needed to provide inocula it is necessary to grow them quickly. They are grown at 18 to 22°C at a distance of 15-20 cm from 65 or 80 W fluorescent lamps, giving a level of illumination at the culture surface of 4 750 to 5 250 1ux (Figure 17). Starter cultures are generally aerated with an air/carbon dioxide (CO2) mixture.
Starter cultures are grown for variable periods of time prior to use. In the case of diatom species, which have short generation times, this period is from 3 to 5 days. For the majority of flagellates it is 7 to 14 days. When ready for use a starter culture is sub-cultured using sterile techniques, as previously described. Twenty to 50 ml, (depending on species and the density of the culture), is transferred to a fresh 250 ml culture - to maintain the starter culture line. The remainder is used as an inoculum for larger cultures (up to 25 l in volume) to be grown for feeding or as an intermediate step in the process of large-scale culture, where they in turn act as the inocula for much larger cultures.
Larger volume starter cultures may be needed to inoculate large-volume production cultures. For clarity, cultures of between 2 and 25 l volume will be referred to as intermediate-scale cultures. As an example, a 200 l production culture will initially begin with a 250 ml starter of the required species which is then transferred when it has grown to a larger volume 2 to 4 l starter. When a 200 l culture is about to be started, 200 to 400 ml of the 2 to 4 l starter culture is used to start a new 2 or 4 l starter culture and the remainder to start the 200 l production culture.
With larger volume starters it is advantageous to increase the level of illumination and to aerate the culture with an air/carbon dioxide mixture. It is advisable to dilute the medium to grow diatom species to a salinity of 20 to 25 PSU (practical salinity units, equivalent to parts per thousand) to obtain the best possible growth rates. Most flagellate species are best grown at about 30 PSU.
3.3 INTERMEDIATE-SCALE CULTURE
Most laboratories and hatcheries requiring small volumes of algae for food use spherical glass flasks or glass or clear plastic carboys of up to 25 l volume (Figure 18). These are generally operated as batch culture systems or semi-continuously. Batch culture involves the inoculation of the culture medium with the required species. The culture is then grown rapidly until a further increase in cell density is inhibited by the failure of the light to adequately penetrate the culture, The culture is then completely harvested, the container washed and sterilized and started again with a new culture.
Figure 18: Two different approaches to the intermediate-scale culture of algae: A - 20 l volume round flasks; B - using equally as effective wine making carboys of 15 to 20 l volume.
The semi-continuous method involves starting the cultures in the same way but instead of completely harvesting them when they have grown, they are partially harvested before the light limiting stage is reached. The harvested volume is then replaced with freshly prepared culture medium and the process repeated 2 or 3 days later. In this way the life of a culture is extended. With some of the hardier species, e.g. Tetraselmis suecica, cultures will last for 3 months or more with harvests of 25 to 50% of the culture volume 3 times each week. Batch culture is generally used for delicate species and the rapidly growing diatoms. Semi-continuous culture is mainly used with hardier species of flagellates.
3.3.1 Growth phases of cultures
Harvesting takes place in semi-continuous culture during the exponential phase of growth. Batch harvests are made generally at the peak of exponential growth as the cultures enter the stationary phase. An illustration of the meaning of these terms is given in Figure 19. In this case the species cultured is the large, green flagellate, Tetraselmis.
Figure 19: Phases in the growth of algal cultures illustrated by a typical growth curve for the large, green flagellate, Tetraselmis suecica.
At inoculation from the starter culture, the starting cell density in the culture is 25 to 50 cells per ml (cells per microlitre). After inoculation these cells grow and divide increasingly rapidly as they acclimatize to the culture conditions. This acclimatization period, which lasts for 2 to 3 days, is called the lag phase. Once adapted to the conditions, the rate of cell division accelerates and increase in the number of cells in the culture is logarithmic. This period lasts for 4 to 6 days and is called the exponential growth phase. Cell division rate then slows as light penetration through the culture and/or nutrients become limiting. The culture then enters the stationary phase, which can last for many days in the case of flagellates or only for a short time for diatoms. Cultures of flagellates remain in this phase by the recycling of nutrients from dead and decaying cells, but in the case of diatoms, which may produce self-inhibiting metabolites, which attract bacterial growth, the culture collapses.
In the example shown in Figure 19, batch cultures of Tetraselmis would be harvested at a density of about 2 000 cells per.l and semi-continuous cultures at about 1 500 cells per.l. These densities can be increased, within limits, by increasing the light intensity falling on the cultures, by maintaining the pH at between 7.5 to 8.2 with controlled CO2 input and by the addition of extra nutrients as the culture density increases.
3.3.2 Details of intermediate-scale culture operation
The complexity of the culture operation depends on the requirement for algae and the cost constraints within which the system needs to operate. In the simplest form the culture system may be just a scaled-up version of the starter cultures, using 2 l to 25 l flat-bottomed, glass flasks or carboys. These are part filled with the culture medium - in this case sterile, nutrient-enriched seawater - and then they are inoculated with the required species and aerated with a mixture of 2% CO2 carried in compressed air. The carbon dioxide is from a bottled gas source with gas pressure and flow regulation. This is to provide the carbon source for photosynthesis and to control pH within the range 7.5 to 8.2. The air/CO2 mixture is filtered through a 0.2.m porosity cartridge or membrane filter to remove the majority of air-borne contaminants and competing microorganisms. Examples of this type of system are illustrated in Figure 18. The culture medium is prepared from filtered or sterilized seawater.
There are various options for culture water treatment:
a) either the seawater is filtered to remove bacteria using 0.22 or 0.45.m membrane cartridge filters, or,b) it is batch or continuously pasteurized at 65 to 75°C or,c) it is autoclaved at 1.06 kg per cm2 for 20 minutes (After autoclaving the medium must be allowed to stand for 2 days in a suitable container closed from the atmosphere). Or,d) it is chemically sterilized with sodium hypochlorite solution at 25 mg per l freechlorine (by adding 0.5 ml of domestic bleach - 5% sodium hypochlorite - per l of filtered seawater). Before use, the residual free-chlorine is neutralized by adding an excess of sodium thiosulphate solution (50.0 mg per l) prepared in distilled water.
Note: Methods (a) and (c) are most commonly used for small-scale culture preparation; (b) and (d), after prior filtration to 1 or 2 µm particle size, for large-scale culture.
After the sterilizing treatment, nutrient additions are made. Details of the nutrient enrichment used at the Ministry of Agriculture, Fisheries and Food, Fisheries Laboratory, Conwy, UK, which is suitable for all of the commonly cultured species, is given in Table 5. Note that diatoms require the addition of silica (Si) to the basic nutrients. The medium is then ready to dispense aseptically to the culture flasks, which are then ready to be inoculated. In recent years, several proprietary brands of algal culture nutrients have become available. These are generally based on the Guillard F/2 formula and provide excellent growth results (see Tables 3 and 4 for the basic formulae).
To obtain the maximum productivity of most species it may be necessary to dilute the seawater with pure (distilled) freshwater (or from an uncontaminated source) before filtration or autoclaving. Growth and cell division rates of Chaetoceros calcitrans, Thalassiosira pseudonana and Skeletonema costatum are optimal at a salinity of about 20 to 25 PSU. Productivity of many of the flagellates is optimal at 25 to 30 PSU.
Illumination for culture growth is provided by fluorescent lamps, usually mounted externally to the culture flasks (see Figure 18). The number of lamps used is determined by the height and diameter of the culture vessels with the object of providing 15 000 to 25 000 lux measured at the centre of the empty culture container. Two 65 or 80 W lamps are sufficient to illuminate 3 l glass flasks, which are about 18 cm diameter, whereas 5 lamps of the same light output are necessary for vessels of about 25 l volume (35 cm diameter). Growth is optimal at 18 to 22°C for most species.
Table 5: Nutrient salt stock solutions for the enrichment of diatom cultures in treated seawater. The addition of stock solution C is omitted in the culture of flagellates.
Examples of cell density achieved in the small-scale culture of a number of nutritionally important species are given in Table 6. These are values obtained at the MAFF Fisheries Laboratory, Conwy, and are typical of densities achieved elsewhere in commercial culture enterprises. It is interesting to note that much higher cell densities of Chaetoceros calcitrans are obtained in 2 l than in 20 l cultures. This does not necessarily mean that productivity in terms of biomass is lower. In all cultured species the size of cells is variable according to culture conditions and the growth phase. In 2 l cultures of Chaetoceros higher cell densities are reached but the individual cells are smaller: 35 µm3 compared with 50 µm3 in 20 l cultures. The dry weight content is also lower at about 10 µg per million cells (micrograms per million cells) compared with up to 18 µg per million cells in 20 l cultures. Other species show similar variability in size related parameters depending on cell density and conditions, quite apart from the inherent differences in cell size between species.
Through manipulation of culture conditions of the larger species, such as Tetraselmis, it is feasible to alter cell size so that the cells can be more readily ingested by smaller larvae. Small-scale culture systems can be technically improved to increase their performance by operating them as chemostats. But, if the objective is solely to produce more food, the better solution is to turn to large-scale culture methods.
Table 6: Cell densities at harvest (cells µl-1) achieved in small-scale batch (B) and semi-continuous (SC) 2 l or 20 l cultures for a selection of nutritionally valuable species. The salinity of the culture medium is also given.
to be continued :