This chapter describes methods used in seedling-rearing stations for culturing young sporelings. Zoospores are collected on substrates (either palm coir “sporeling ropes” or bamboo rod “chopsticks”) where they develop into gametophytes. Male and female gametes produced by gametophytes combine sexually to form zygotes which develop into sporelings. The gametophyte generation is completed in about twelve days. Sporelings attached to sporeling ropes are cultured in the seedling station for a period of three months. After reaching a length of 2–5 cm the sporeling ropes are transferred to the raft site for intermediate culture
Since the early 1950's two basic methods have been used for Laminaria sporeling culture: (i) outdoor sporeling culture under natural conditions in seawater, used between 1949–1956, and (ii) indoor sporeling culture under artificially controlled conditions in a seedling-rearing station, used after 1956. The artificial seedling-rearing method of sporeling culture may also be subdivided into two methods: (a) seedling-rearing with an artificial light source (fluorescent lamps), used between 1956–1958, and (b) seedling-rearing with natural daylight (a glass house), developed after 1958.
Natural Sporeling Culture in Seawater
In the early 1950's sporeling culture was done outdoors in shallow sea regions. Spores were collected in mid-October, when seawater temperature drops to 20° C, by lowering bamboo substrates into parent Laminaria beds. The bamboo substrates, flat bamboo sections about 0.5 m in length and 10 cm in width, were tied together in series like rope ladders (Fig. 3.1). They were anchored in place and buoyed with glass floats. Zoospores released into seawaters by parent kelp plants attached to the bamboo substrates and developed into “autumn sporelings”. After about three months of growth the young sporelings were transplanted to thicker culture ropes which were suspended from floating raft ropes for the final grow-out period.
Artificial Sporeling Culture in Seedling Stations
After 1956 a new “summer sporeling” culture method was developed to overcome many of the problems encountered with “autumn sporeling” culture. Under this new method, now widely practiced in China, zoospores are collected in mid-July before seawater temperatures rise above 20° C and are cultured indoors in seedling stations using artificially cooled seawater. The zoospores are induced to adhere to artificial substrates such as bamboo rods or palm-fibre seedling cords. The substrates are submerged in culture tanks through which cooled water is circulated. Water temperature is maintained at between 8–12°C, preferably 8–10°C, throughout the seedling-rearing process. After three months of growth in the seedling station (mid-July to mid-October) young sporelings reach a length of between 2–5 cm.
Fig. 3.1. Bamboo rod substrates used for collecting zoospores from naturally growing parent Laminaria beds.
a: connecting rope b: bamboo rods c: stone weight
In mid-October when seawater temperature falls to about 20° C the summer sporelings grown to a length of 2–5 cm in seedling stations are transferred to raft ropes in natural seawaters. Following an intermediate culture period of 2–4 weeks (mid-October to mid-November), during which sporelings grow in length to between 10–25 cm, young sporophyte plants are ready to be transplanted from the sporeling ropes to thicker culture ropes which are suspended from floating raft ropes for the final growout period. The grow-out period lasts until harvest in mid-July of the following year.
As discussed in Chapter I, the use of artificial seedling-rearing techniques for growing summer sporelings lengthens the growing season by 2–3 months and increases yield by 40%, compared with methods previously used for culturing autumn sporelings.
Artificial Seedling-rearing Using Natural Daylight
Seedling stations constructed in the mid-1950's were closed buildings in which lighting was provided by fluorescent lamps. In the late 1950's “glass house” seedling stations were constructed so that natural daylight could be used instead of fluorescent lamps, thus saving on electricity costs. The refinement is sometimes referred to as “industrial sporeling culture under daylight”. As in agricultural greenhouses, illumination is controlled in the seedling-rearing “glass houses” with curtains that can be opened or closed depending on light exposure required. As before, seawater is artificially cooled to stimulate the growth of young sporelings.
The first seedling station designed to use natural daylight was built in Shandong Province in 1958. Its glass-enclosed culture room had a total area of 5,200 m with a production capacity of 400 million sporelings per year, highest in the world for such a facility. Today the new design has become an industry-wide standard.
The seedling-rearing station is composed of two main systems: (i) the sporeling culture room, and (ii) the water circulation system. The former system includes the glass house and culture tanks used for controlling illumination and for sporeling culture. The latter system includes: (a) the filtration system for purifying seawater, (b) the indoor water circulation system for pumping seawater through the culture tanks.
Site Selection for the Seedling Station
Successful sporeling culture depends on such factors as seawater quality, daylight exposure and building design. Site selection must consider these and other factors which determine operational efficiency. The following are important criteria for site selection:
The station should be located on the shore near seawater so that pumping costs are reduced to a minimum.
The station should be located far away from any sources of water pollution, such as harbours, estuaries, industrial or residential areas.
The station should be located at a place where the nearshore seafloor is sandy or rocky so that pumped seawater will not contain excessive amounts of mud or organic debris.
The station should be located in open terrain where there are no trees or high buildings to block sunlight or impede air circulation.
The station should be located on a level site where ground is firm, avoiding sandy beaches and regions with soft soils, in order to provide a solid foundation for the building structures.
The station should be located at a place where there are good utilities and transportation services, i.e. where there are good roads, a good power supply and a good supply of freshwater.
General Layout of the Seedling Station
The design of the main building structure must be economical and must suit particular conditions found at the building site. The sporeling culture room containing the culture tanks, as well as the other associated structures (a pumping station, water filtration tank), must be built on firm and level ground. Piping systems should be buried underground.
Layout of the Sporeling Culture Room
The sporeling culture room, or “glass house”, is the main structure of the seedling station, where the culture tanks are placed for raising sporelings. The typical sporeling culture room, designed for production of 400 million sporelings annually, is constructed like a greenhouse. The interior area of the culture room, usually about 5,200 m2, is enclosed with a glass-pane roof. All open spaces of the culture room should be equally exposed to bright daylight, with no supporting walls or other shadow-making fixtures blocking incoming light.
The four outer walls of the “glass house” or “culture room” should be made of reinforced concrete rising about 60 cm above ground level. The inner floor area of the culture room, made of poured reinforced concrete, should be designed so that there is a gradient of levels, like a series of steps, each floor level designed to hold a row of culture tanks (Fig. 3.2). The glass roof of the station should be positioned to face east-west in order to receive highest daylight exposure. Glass roof sections imbedded in a metal frame are raised over the culture room. Glass walls are constructed between the cement foundation and the glass roof structure. The roof may be formed of one or two layers of glass depending on the degree of insulation required for maintaining stable indoor temperature.
Simple but effective systems must be installed for adjusting air circulation and light intensity. Indoor window shutters should be built along the glass walls. Moveable curtains should be fixed in place below the glass roof. Light intensity may be further controlled by painting the exterior surfaces of the glass roof with light-reflecting white paint. Methods for adjusting air circulation are required, such as hinged windows in the glass walls and circulation fans.
Building materials should be strong, durable and noncorrosive. In China, construction teams often use prefabricated reinforced concrete components that can be welded in place, especially for framing main structural elements such as roof beams, supporting pylons and window frames. Materials such as wood and iron which decay or corrode quickly when exposed to seawater or high humidity should be avoided as much as possible.
Fig. 3.2. Layout of seedling station culture room.
Showing the glass-roofed culture room with culture tanks arranged on different floor levels to enable a gravity-fed water circulation system.
If the culture room is built on sandy subsoil a reinforced concrete retaining wall should be constructed around its outer periphery to prevent shifting of the subsoil. Highly reinforced concrete should be used in floor areas that will support the weight of culture tanks to prevent cracks and seepage of seawater.
Culture tanks are made of reinforced cement, their dimensions depending on their arrangement in the culture room. Culture tanks are usually about 8–10 m in length, 2.15–2.3 m in width and 0.3 m in height. The tanks should be designed so that they fit in rows on the gradient of floor levels in the culture room. This system allows deployment of a gravity-fed water circulation system, enabling seawater to flow from one row of tanks placed on an upper level of the culture room to another row of tanks placed on a lower level (Fig. 3.2).
If there are six or fewer levels across the open floor area of the culture room and if 20% of total seawater in the water supply system is changed daily, the water temperature differential between entry and exit canals of the culture room should be less than 2° C.
Layout of the Water Circulation System
The water circulation system consists of the intake pipe, the settling tanks, the pumping stations, the filtering tanks, the holding tanks, the water refrigeration system and the piping system which circulates recycled seawater through the indoor culture tanks (Fig. 3.3).
Seawater is pumped through an intake pipe into large settling tanks. The end of the intake pipe submerged at sea is surrounded by a wire cage to prevent intake of debris. The settling tanks are used for precipitating out any mud and other suspended debris or solids, prior to filtering.
From the settling tanks the seawater is pumped into large scale filtering tanks which contain layers of gravel, fine sand and activated charcoal. Seawater pumped into the top of the filtering tanks percolates through the filters, removing colloidal particles, fine silt and microorganisms. The purified water is then pumped into elevated holding tanks.
Seawater is immediately pumped through inflow canals to the upper level culture tanks in the culture room. The water flows from higher level culture tanks to lower level culture tanks, exiting the culture room through outflow canals. Sea water exiting the culture room is pumped through a special filtering tank (bypassing the settling tanks and water treament) to be recycled through the indoor water circulation system of the culture room. Recycled seawater is periodically and systematically renewed by being replaced with freshly filtered and returned seawater.
Fig. 3.31 Water circulation system of the seedling-rearing station.
1: wire cage 2: seawater intake pipe 3: cement support 4: pumping station 5: settling tanks 6: filter tanks 7: watermixture tank 8: glasshouse 9: recycled water circulation system 10: waste water outlet pipe
In Fujian Province the indoor gravity-fed circulation system is sometimes supplemented with electric stirring machines that increase water flow through the culture tanks, so that effective cooling in the culture room is maintained in a warmer climate.
The process of seedling-rearing begins with zoospore collection in mid-July. Zoospores are collected from parent Laminaria stock specially selected for this purpose. To guarantee good production of healthy zoospores, the raising of parent Laminaria stock is often entrusted to companies which specialize in this undertaking or is done by work units directly engaged in seedling-rearing. The practice of specializing in the raising of parent Laminaria stock for zoospore production has the following advantages:
all activities relating to seedling-rearing can be well-planned and coordinated;
robust parent stock can be separated from general raft culture production and specially cultured in very fertile waters using most appropriate grow-out management techniques;
selected strains may be used for long term interbreeding programs, creating hybrid varieties that have desired characteristics, such as higher production rates, higher iodine, mannitol and alginate content and higher resistance to warm seawater temperatures.
Selection of Sea Region for Culturing Parent Laminaria
Sea regions selected for culturing parent Laminaria stock should have the following characteristics:
Fertile water. With dissolved nitrate and phosphate salts in concentrations of at least 20 mg and 5 mg per m3 respectively.
Good water exchange. Water should be clear and moderately fast-flowing, indicating good gaseous exchange and adequate inflow of nutrients.
Moderate transparency. Stable water transparency not only promotes even exposure of kelp plants to sunlight, but also prevents white rot and green rot diseases caused by widely fluctuating water transparency.
Low levels of silt and epiphytes. The seawater should have low levels of suspended mud and silt. Seawater should also be relatively free of epiphytic seaweeds and attaching organisms which cause frond deterioration, block photosynthesis and inhibit development of smooth clean blades capable of producing healthy sporangial sori.
Calculation of the Number of Parent Laminaria Required
A hatchery designed for production capacity of 400 million young sporeling plants annually will require 8,000 to 10,000 parent Laminaria plants for spore collection. i.e. each parent plant is calculated to produce 40,000 zoospores. In order to guarantee sufficient quantity and quality of parent Laminaria plants, at least twice this number should be cultivated. In other words, 16,000 to 20,000 parent kelp plants should be grown to achieve the given production target of 400 million young seedlings.
Cultivation methods used for growing parent stock differ from normal raft culture methods. In normal kelp culture high density planting is practiced to maximize yield at harvest. The objective of parent Laminaria cultivation, however, is to grow high quality plants with broad thick fronds that will develop abundant sporangial sori.
Therefore culture of parent stock requires that plants should be spaced at wide intervals on shorter kelp culture ropes. This “thin cultivation” or “low density” cultivation method, together with careful selection of seawater regions, creates optimum growing conditions for culturing healthy parent stock for zoospore collection.
Low Density or Thin Cultivation Method
Parent Laminaria sporelings should be selected from only the most robust of “first batch” intermediate culture sporelings. The young sporelings should have fronds that are wide, thick, smooth and lustrous. They should be transplanted in low density to short kelp culture ropes, spaced at 5 cm intervals on culture ropes 80 cm in length, with 16 plants per culture rope. Transplantation to culture ropes should be carried out as early as possible in order to lengthen the grow-out season.
Kelp ropes with attached parent Laminaria should be suspended from parallel floating raft ropes in the usual “hanging kelp rope raft culture” method. Kelp ropes should be submerged to a depth at which top plants on culture ropes are no deeper than one metre below the water surface. As usual, bottom ends of hanging culture ropes should be weighted with stones. Culture ropes should be spaced 50 cm apart, with an overall cultivation density of approximately 4,000 parent kelp plants per mu (60,000 per ha).
Grow-out Management Procedures for Parent Laminaria Stock Adjustments of Water Depth
The main tasks to be performed during grow-out management of parent Laminaria stock are: (i) adjusting the water depth of culture ropes during different grow-out stages, (ii) reversing hanging culture ropes to equalize illumination on all plants, and (iii) periodic washing and cleaning of the plants to remove silt, epiphytes and other attaching organisms.
Transplantation of parent plants to culture ropes is carried out between mid-to-late November. For a period of time following transplantation plants are light sensitive and so the tops of kelp culture ropes should be submerged at a depth of 1 m to lower illumination levels. In late February or early March the kelp culture ropes should be reversed one time. Between late March and early April the culture ropes should be gradually raised in the water to a depth of 50–60 cm. Raising kelp ropes increases illumination, thereby improving the growth and development of broad thick fronds with good accumulation of photosynthates. Blade tips of maturing plants may be cut during the latter half of April to improve illumination and reduce frond deterioration. Around mid-June the kelp ropes should be lowered to a depth of 70–80 cm in order to shade plants, since sporangial sori form best in dim light conditions.
In general sporangial sori are first formed on the shady side of the blade, whereas the side facing the light develops sori more slowly. Nevertheless, maturation of sporophytes and extent of formation of sporangial sori are closely related to water temperature (Fig. 1.6) and light intensity. Experimental evidence reported in Fig. 1.13 indicates that sporangial sori form best on mature plants cultured at shallower depths of 50 and 100 cm, where light intensity is higher. Table 3.1 shows that Laminaria fronds cultured at shallower depths mature earlier and the proportion of plants bearing sporangial sori is higher, since they receive higher light exposure. There is no appearance of sporangial sori on plants cultured at lower depths even by mid-May when seawater temperature has risen to 17.1° C, a temperature within the range of 15–10° C is most favourable for sporangial sori formation (Fig. 1.6).
The area of blades developing sporangial sori is also closely related to light intensity. Fig. 3.4 shows results of a study of sporangial sori formation on plants cultured at different depths of 60, 100 and 200 cm. Although temperature range and nutrient levels were optimum, results of the study indicate that deficient illumination at lower depths results in a decreased area of formation of sporangial sori on blades. All measurements were made for blades of equal size. The results again indicate that formation of sporangial sori depends on light intensity and light duration.
Fig. 3.4. Rate of formation of sporangial sori on mature Laminaria blades grown at different depths. Liu Tianjin, 1979.
Cleaning of Fronds
Good management requires that plants should be cleaned periodically during grow-out. This is done by shaking the kelp ropes vigorously (without shaking plants loose) to remove settled deposits of mud and silt. This procedure improves photosynthesis and gaseous exchange, thus enhancing growth. Occasionally kelp blades may need to be cleaned more thoroughly to remove any epiphytic seaweeds or other attaching organisms clinging to the surfaces of the fronds. This is done by lifting and hand-rubbing individual blades, taking care not to injure blade surfaces.
Providing Fertilizer Additives
If there are insufficient levels of dissolved nutrients in the seawater, fertilizer application is required. This is done using either the hanging bag method (where fertilizer is placed in plastic bags and diffuses through small pinholes into the seawater) or the sprinkling/spraying method (where fertilizer in low concentration solution is sprinkled by hand or sprayed with high-pressure hoses over the seafarming area). Generally, nitrogen-based fertilizer is applied at the rate of 150–250 kg/mu (2,250–3,750 kg/ha) apportioned over the 6–7 month grow-out period.
Selection of Mature Parent Laminaria
Selection of high quality parent Laminaria is done when sporangial sori are seen forming on the fronds. Selection should take place towards the end of June in northern China, i.e. before the general harvest in mid-July. (Where parent plants are selected from rafts used for general production.) Selection should be scheduled to take place about 20 days before planned timing of zoospore collection.
Best parent Laminaria plants are those with large areas of sporangial sori, especially along the main mid-band of the blade. Plants should have thick and wide blades that are pliable and stout, with a deep lustrous colour and with a strong stipe, and should be free of epiphytes and other clinging organisms. Blades should have no patches of rot or deteriorating edges.
Selected parent Laminaria plants are removed from culture ropes and transported to shallow water. First they are trimmed to remove parts of blade tips and edges showing signs of deterioration or having poorly developed sporangial sori. The tangled rhizoid is pruned, leaving only the main or primary rhizoids. The fronds are then carefully cleaned and washed to remove attaching organisms, epiphytes and silt.
After trimming, plants are reattached to culture ropes, 15– 20 plants being tied to each 1 m long culture rope. The culture ropes are again suspended from rafts at a depth of 1 m in clear seawater with moderate currents and free of silt. In this way parent Laminaria plants are temporarily cultivated to stimulate further maturation of sporangia and to allow recovery from the trauma of pruning. During this temporary stocking and cultivation period, plants should be washed periodically to prevent accumulation of debris and silt on blades which would impede development of sporangial sori.
Oversummering of Parent Laminaria in Southern China
In northern China, as described above, parent Laminaria stock may be selected directly from mature plants growing on rafts in the general production area. Selection of parent plants is done towards the end of June. After a period of temporary cultivation spores are collected in mid-July. The spores are then cultured into young sporelings under artificial conditions in seedling-rearing stations.
In southern China, for example in Fujian Province, if parent Laminaria stock were left over the summer in the seawater they would be severely damaged by high summer seawater temperatures. Therefore a different procedure for cultivating parent Laminaria stock has been adopted, called “oversummering of parent stock” (Fig. 3.5). Oversummering prevents blade deterioration caused by high seawater temperatures and thus conserves healthy parent stock for use in zoospore collection in mid-September. The “oversummering” procedure has two stages, with parent stock being selected on two occasions:
In early June, before seawater temperature rises above 20°C, parent stock is selected from general raft production plants. Selected culture ropes with chosen parent plants are moved to a temporary location for continued growth. The location chosen should be a place where water is clear and where current flows are active. Here special rafts are stationed for the purpose of temporary cultivation of parent stock.
In early July, when seawater temperatures rise to about 26° C in southern China (a temperature approaching lethal levels for sporophytes), parent stock is again selected from plants at the temporary growing site. These plants are moved to indoor culture tanks in a seedling station where they are grown throughout the summer months in artificially cooled water. The procedure is known as “oversummering” of parent stock.
In this way parent stock is conserved, protected from exposure to damaging effects of high summer seawater temperatures that would be suffered without the oversummering procedure.
The Oversummering Procedure
Tips and edges of selected plants are pruned away, leaving only a portion of mid-blade 60–70 cm long and 20 cm wide. After washing and cleaning, the pruned parent blades are transferred to indoor tanks in the seedling station, with 4–5 kelp plants occupying each square metre of tank space. Here the plants are oversummered in cooled water at 8–10° C and under controlled illumination at 3,000–4,000 lux. One-third of the water in the tanks should be renewed with freshly filtered seawater each day. The cooled seawater should be recycled through a water circulation system for at least 16 hours daily.
Fig. 3.5. Outline of procedures used in Southern China
for culturing parent Laminaria stock.
After 20 days of cultivation under these controlled conditions, i.e. around mid-July, sporangial sori will begin to form on the blades. After another 20 days, when the epidermal layer of the sporangial sori ruptures, light intensity in the seedling station should be reduced to 700–1,000 lux. Also, the water temperature should be raised gradually 0.5–1.0° C every 3 days to 13° C. After 50–60 days of artificial cultivation, i.e. towards the end of August, zoospores are ready to be collected from mature sporangia.
Oversumering parent plants in cooled seawater delays maturation of sporangia on parent fronds. Thus spore collecting can be postponed up to three months. Hence the period of seedling-rearing can be shortened by up to 80 days, resulting in a very significant saving on production costs.
A substrate is the material on which motile zoospores settle and attach. Laminaria spores will attach to a wide variety of substrates, both natural and artificial, including rocks and boulders, shells, wooden rods, bamboo poles, metal objects and strands of rope.
Substrate materials used in seedling stations must be lightweight and easy to handle, have large surface area and be free of toxic substances that would affect water quality. The two most frequently used substrates are: (i) natural palm fibre rope and (ii) bamboo rods.
Palm Fibre Ropes
Palm fibre rope is mainly used in northern China as well as in Fujian Province. It has high tensile strength, shows good resistance to rotting, has a large surface area per unit length, is easy to handle and does not exude any toxic substances. There are many types and grades of palm rope. Palm rope aged to a dark reddish or rust-brown colour works much better than palm rope whose fibres are light green in colour. Freshly stripped green fibres tend to release a sticky sap which is lethal to young sporelings.
Palm rope should be prepared using carefully chosen palm fibres. After removing the bark and outer skin of palm branches, palm fibres at least 20 cm in length should be stripped and gathered for spinning into palm rope. This is done using a special rope-spinning machine. The final twisted strands of palm rope substrate should be about 0.5 cm in diameter.
Bamboo Rods: Chopsticks
Floating mud and silt tend to adhere to palm fibre rope, whereas bamboo rods, which have hard, smooth, shiny surfaces, prevent such accumulation. Therefore bamboo rods are used as a substrate material for zoospore collection in southern China where muddy deposits in seawater are relatively high.
So-called “bamboo chopsticks” are slender bamboo rods that have been split from mature bamboo poles. Each rod should be about 35 cm long, 0.9 cm wide and 0.5 cm thick. Older bamboo at least six years in age is best because it is harder and doesn't exude as much fresh sap. Also older bamboo with a hollow core is better than younger bamboo with a solid core, because the former can be split more easily into chopsticks. During splitting it is best to make chopsticks which are rectangular or flat in shape with a wide shiny outer surface area.
Preparing Palm Rope for Use as a Substrate
All materials used for substrates must be thoroughly cleaned to remove harmful organic compounds, especially tannic acid. Procedures for processing and cleaning palm fibre rope are as follows:
First, the newly twisted coir rope must be made pliable. This is done by “dry hammering”, where the rope is pounded with a special hammering machine. Each section of palm rope should be hammered about 400 times. The process removes all remaining palm bark and other unwanted fragments. It also makes the palm rope very flexible and thus easy to handle.
After soaking, the palm rope should be pounded again in a procedure called “wet hammering”, which uses the same hammering machine as in dry hammering. Each section of rope must again be pounded about 400 times. During hammering the rope should be sprayed with freshwater to wash away any exuded substances.
After wet hammering the rope should be boiled in large vats for 3–5 hours and left soaking in the water overnight. Finally the rope is washed in clean freshwater and dried in the sun.
Preparing Bamboo Rods for Use as a Substrate
The preparation of bamboo rods is even more stringent than preparation of palm ropes. First, the rods should be soaked in a 1% alkaline seawater solution for 30 days, changing the alkaline seawater solution every 10 days. Then the rods should be sterilized by boiling them in vats containing a 1% alkaline seawater solution, with the water temperature kept above 80° C for 24 hours. After boiling, the rods should be immersed in flowing water for 7–10 days to remove all impurities. Finally, the bamboo rods should be spread over a clean outdoor ground area for sun-drying.
Making Sporeling Curtains
“Sporeling curtains”, also called “culture mats”, are made from the cleaned substrate materials and are used in seedling stations for rearing young sporelings. They are so-named because they look somewhat like loosely draping window curtains or woven floor mats.
The palm rope sporeling curtain is constructed in one of two ways, either (a) with palm ropes hanging freely between wooden or bamboo end–pieces, or (b) with palm ropes woven inside a fixed frame (Fig. 3.6: a,b).
Dimensions of culture mats depend on dimensions of culture tanks used in the seedling-rearing station. The length of culture mats should be slightly shorter than the inside width of the culture tanks, so that the finished mats can be easily spread inside the tanks. Mats are usually rectangular in shape, about 1.25 m in length and 0.45 m in width.
The culture mat consists of a long length of palm rope wound between a wooden frame. Materials used for constructing the frame must be carefully sterilized in boiling water. About 25 equally spaced small nails are fixed into each end of the frame so that the palm fibre rope can be wound around them to form the culture mat. For a wound culture mat 1.25 m in length and having 50 palm ropes woven end-to-end between its frame-ends, the total length of palm rope required is: 50 × 1.25 m = 62.5 m. The coir rope should be formed into balls and soaked in clean water to improve its flexibility and ease of handling during the weaving of culture mats. The end-to-end lengths of rope woven within the culture mats are referred to as “sporeling ropes”.
After the palm rope has been wound on the frame, 5–6 additional smaller fibre cords should be woven in-and-out across the width of the culture mat in order to strengthen the mat and to keep the lengthwise ropes separated and evenly spaced (Fig. 3.6:3). Each completed mat can hold about 50,000 attached sporelings.
Loose palm hairs on culture mats may cause problems since young sporelings may easily detach from them. Therefore loose hairs should be removed by wetting the palm rope curtains and placing them for a short time on hot charcoal coals. This singes and burns off any straggling hairs.
Fig. 3.6. Palm rope sporeling curtains or mats.
a: palm ropes freely suspended between wooden end pieces
b: palm ropes woven within a fixed wooden frame
|1: end frame||2: palm rope cords|
|3: cross ropes||4: fixed side frame|
Bamboo Culture Boards
About ten bamboo chopsticks are tied tightly together, with all shiny surfaces facing in one direction, to form a “culture board”, 35 cm long, 9 cm wide and .5 cm thick. Pairs of bamboo culture boards are tied back-to-back, with their shiny surfaces facing outward. Rows of culture boards are then suspended within a hinged wooden frame whose length fits inside the width of culture tanks being used (Fig. 3.7). Many frames are placed inside each culture tank. In this way a large number of culture boards for zoospore collection can be immersed in a relatively small volume of culture tank space. The frame holds the culture boards at their, top and bottom ends and is hinged so that it can support the culture boards vertically or in a slanted position. This allows frequent adjustments to increase illumination in the spaces between the suspended culture boards. The method is used only in a few seedling stations in Zhejiang Province in southern China.
Fig. 3.7. Bamboo culture boards suspended in a culture tank. (Overhead view.)
a: pair of culture boards, each culture board 9 cm
wide consisting of 10 bamboo chopsticks tied together
b: supporting wooden frame
Spore collection is equivalent to sowing of seeds in land crop farming. Briefly, the process involves partial drying of parent Laminaria fronds to stimulate release of spores from sporangial sori. After drying stimulation the parent fronds are placed in a small volume of seawater in the indoor culture tanks, together with the substrate materials already described. Liberated spores attach to the substrate materials - either palm fibre seedling cords or bamboo chopsticks. These “seedling ropes” or “sporeling ropes” are then placed in other culture tanks for the duration of sporeling cultivation.
Timing of Spore Collection
Laminaria sporophytes cultured on rafts mature and release zoospores between April and mid-July in northern China. Zospore collection is timed to take place as late as possible, i.e. in mid-July before summer seawater temperature rises above 20° C. If zoospores were collected earlier, the period of cultivation in the seedling-rearing station would have to be extended, thereby raising production costs. A longer cultivation period would also mean that sporeling growth would have to be inhibited, otherwise young seedlings would have to be transplanted too early to the grow-out rafts. Inhibiting sporeling growth would be both complex and costly. On the other hand, if zoospore collection is delayed too late, midsummer seawater temperature will rise above 21° C. Above 23° C relatively few zoospores will be produced and those that are produced will not be vigorous (Fig. 1.6).
Collecting Zoospores and Diluting Spore Density
The parent Laminaria fronds are placed carefully in a spore collecting tank where seawater temperature is 8–10° C. Almost immediately sporangia on the mature fronds rupture and release spores. The fronds should be shaken periodically to stimulate spore release. Water samples should be checked under a microscope to observe the density and vigour of the motile zoospores. Healthy spores swim quickly and move in straight lines in the seawater, whereas weak spores swim slowly and move in circles. Only strong spores should be counted. When the spore count reaches 15–20 per field area at 100x magnificacation under a light microscope, then spore density is optimum for adhesion to substrate materials.
A typical standard parent Laminaria plant will have an area of 800 cm2 of sporangial sori on its frond surface. Between 80– 100 standard parent plants are used per cubic metre of water for collecting zoospores. The spore density in the seawater will reach the required 15–20 spores per 100x field area within 1–1/2 to 2 hours. A good quality parent plant may be used for spore release 2–3 times in several spore collecting tanks over a period of 4–6 hours.
Another method is to allow spore density to exceed the required 15–20 spores per 100x field area. This is done by leaving parent fronds in the collecting tank for 4–6 hours. Then the seawater containing a high density of spores can be diluted to the required level for spore adhesion purposes. This is done either by siphoning seawater from the spore collecting tank into other tanks containing seawater, or by adding more seawater to the spore collecting tank. Dilution should continue until spore density reaches a level of 15–20 spores per 100x field area.
Spore Attachment to the Substrate Materials
Generally, 5–7 layers of palm rope culture mats are positioned in one tank in preparation for spore attachment. After spore adhesion takes place, the layered mats will then be distributed to 5–7 other tanks for sporeling cultivation. (Thus 1/5th to 1/7th of the culture tanks in the seedling-rearing station are used for the spore collecting procedure.) The layered mats or curtains are separated with supporting blocks of wood so that seawater can circulate between them. With the culture mats in place, seawater containing zoospores is siphoned into the tank, care being taken that all surface areas of the substrate materials are well-submerged.
The density of spores attaching to the surface area of substrate materials is critical for sporeling development. If density is too high, development of young seedlings will be impeded due to overcrowding and their survival rate will be greatly reduced. The best density of spores per surface area of substrate is about 20–50 spores per 100x field area. If spore density is higher or lower than this range, then the rate of sporeling growth on artificial substrates will be decreased (Table 3.2).
Glass microscope slides are used to measure the density of spores adhering on substrates. The slides are immersed in seawater near the substrate materials and spores adhere to them at the same rate that they adhere to the palm rope seedling curtains.
The motile spores take 3–5 hours to settle and adhere to the substrate materials when the seawater temperature is between 8– 10° C. The rate of settling and adhesion can be periodically checked by sampling the glass slides and counting the number of attached spores under a microscope. Spore adhesion should be stopped as soon as the density of attached spores reaches 20–50 per 100x field area by quickly transferring the substrate materials to other culture tanks which have been prepared with fresh seawater.
Arrangement of palm rope seedling curtains in the culture tanks is an important factor determining sporeling production rates.
i. Flat Plane Arrangement
The flat plane arrangement is used mainly in Shandong and Liaoning Provinces in northern China. Culture mats used are of the loose-hanging type, i.e. sporeling cords freely suspended between end-pieces rather than being woven within a fixed frame (Fig. 3.6a). Loose-hanging sporeling curtains are easily moved about, especially during cleaning operations (described below).
Substrate curtains are laid flat, submerged about 8–10 cm beneath the water surface in the culture tanks. They are also raised 8–10 cm from the bottom of the culture tanks by being held on a supporting frame or on supporting ropes. The frame, fitted to the sides of the culture tanks, is made of wooden or bamboo poles spaced 2 m apart, with polyethylene ropes (diameter 0.4 cm) tied lengthwise at 20 cm intervals between the poles (Fig. 3.8). A simpler method is to use two ropes fixed end-to-end in the culture tanks and raised 8 cm from the bottom of the tanks. Culture mats are laid on top of these supporting structures, whose purpose is to allow circulating seawater to flow freely beneath and above them.
Fig. 3.8. Flat plane arrangement of culture mats.
a: top view showing culture mats placed in flat position on supporting ropes in culture tank
b: side view showing culture mat raised on supporting ropes in culture tank
1: supporting pole 2: supporting ropes 3: single culture mat or curtain made of palm rope
The flat plane arrangement of culture mats has several important advantages. Firstly, transformation from spores into sporelings is very successful, with higher survival rates compared to alternative methods. Secondly, all sporelings grow evenly because they are exposed to equal light intensity. Thirdly, this arrangement offers best advantages for efficient operation and management of sporeling culture, with lower manpower and production costs.
ii. Inclined Plane Arrangement
The inclined plane method of arranging culture mats is used chiefly in Fujian Province in southern China. Culture mats are formed of palm ropes woven within a fixed frame (Fig. 3.6b). Two culture mats are leaned against one another, with their top edges tied together and their lower edges spaced about 17–18 cm apart. A stone weight is suspended from the top edges of the frames between the two inclined culture mats to exert downward stabilizing force. About 10–15 pairs of inclined culture mats can be arranged in rows in each culture tank, depending on the size of tanks being used (Fig. 3.9).
Fig. 3.9. Inclined plane arrangement of culture mats.
a: top view of culture mats in the culture tank
b: side view of pairs of inclined culture mats arranged in a row in the culture tank
1: pairs of inclined culture mats 2: weight
The inclined mats are immersed in seawater in the culture tanks. The depth of seawater in the tanks is therefore greater than for the flat plane arrangement of culture mats, thus realizing more efficient utilization of the culture tanks.
A strong disadvantage of this arrangement, however, is that sporelings on inclined culture mats receive uneven exposure to light, sporelings on upper parts of mats receiving more light than those on lower parts. Consequently there is a difference in growth rates, sporelings on upper parts of inclined culture mats growing larger and more quickly than those on lower parts.
Sporeling culture on artificial substrates in seedling stations is highly intensive in nature. Multiple factors affecting production of young sporeling plants must be carefully controlled. Some of the most important technical considerations for high intensity production of young sporelings are the following:
i. Water Temperature
Optimal temperature ranges for the development of male and female gametophytes differ, the former developing best in the range of 10–15° C, the latter in the range of 15–20° C (Tables 3.3 and 3.4).
Completion of the gametophyte generation with formation of zygotes takes an average of 13.5 days at 10° C. Whereas at 5° C or 15° C completion of the gametophyte generation takes an average of 16 days (Table 3.5). Therefore water temperature in the seedling-rearing station during the first two weeks after zoospore collection should be maintained at a temperature of about 10° C.
For the remaining period of sporeling growth in the seedling station, water temperature should be maintained in the range of 8–10°C, with a maximum of 12°C.
Temperature of natural seawater near the seawater inlet to the settling tanks should be measured daily. The temperature of seawater entering the indoor circulation system, as well as the indoor temperature of the culture room, should be measured hourly. The temperature of seawater at the exit drain from the indoor circulation system should be measured every eight hours.
ii. Light Intensity and Periodicity
Seaweeds are autotrophic, depending on good light exposure for promoting photosynthesis. Control of light exposure includes management of both: (a) light intensity (rate of exposure) and (b) light periodicity (duration of exposure).
Release of zoospores can occur with or without illumination. However under weak illumination zoospores exhibit obvious phototaxis. Whereas under strong illumination zoospores tend to move away from the light. The fact that embryospores germinate either in total darkness or in light intensity varying from 50– 4,000 lux shows that light has no effect on germination of embryospores.
During gametophyte growth optimal light intensity is around 1,000 lux. Illumination below 200 lux may result in slow growth or may impede growth entirely (Table 3.6).
Sporelings require different light intensity in different growing stages. From zygote formation to the time that sporelings reach a length of 0.1 cm in length, optimum light intensity is 1,000–2,000 lux. Between 0.5–1.0 cm, light intensity should be increased to 2,000–3,000 lux. And sporelings between 1–2 cm in length require relatively strong illumination of 3,000–4,000 lux. About 10 hours of daylight is sufficient for sporeling growth in early developmental stages (Table 3.10).
Daylight illumination changes with the weather. Fluctuations of daylight intensity in the culture room should be carefully monitored. Light intensity measurements should be recorded hourly or when any noticeable weather changes occur. The results should be compiled in a monthly summary record. Patterns of change in daylight intensity may thus be observed, enabling regular adjustments of illumination to desired levels.
Natural daylight intensity frequently exceeds the level of light intensity required for optimum sporeling growth. Therefore daylight intensity must be controlled in seedling-rearing stations by: (a) applying white paint on the inside surfaces of the glass roof and (b) using indoor moveable curtains installed below the roof windows. Daylight intensity should be tested frequently in the culture room, usually at hourly intervals.
Even distribution of illumination on sporelings can be further controlled by rotating sporeling plants in the culture tanks on a regular basis. This can be done during the daily cleaning of sporeling mats.
iii. Water Quality
Measuring water quality is also part of the routine management of sporeling production in a seedling-rearing station. This includes taking measurements for: turbidity or transparency, specific gravity, salinity, acidity (pH), and dissolved concentrations of gases and elements (oxygen, carbon dioxide, nitrate-N, ammonia-N and phosphorous). Generally, these measurements should be taken every 3–5 days. Following rainstorms the specific gravity and salinity of seawater entering the indoor circulation system should be tested. Levels of dissolved ammonia-N should be checked at least once daily so that any problems arising can be caught quickly. Standard values for some of the main factors of seawater quality affecting growth of Laminaria sporelings are given in the following table:
|standard values of main factors|
iv. Nutrient Requirements
Conditions in sporeling culture tanks differ from natural sea conditions in several ways. Firstly, water in culture tanks is calmer, much less agitated than natural seawater which is buffeted by tide and wave action. Secondly, the density of sporelings in culture tanks is greater than sporeling density on natural substrates in seawater. For these reasons it is important that nutrients be replenished when sporelings are cultured intensively under artificial conditions in indoor culture tanks.
Levels of dissolved nitrogen and phosphorous in the seawater circulating through culture tanks have no obvious effects on embryospore germination, but do affect growth and development of gametophytes. Under experimental conditions, if phosphate levels are elevated and nitrogen is removed from seawater, gametophytes continue growing and show egg extrusion but their rate of development is very slow (Table 3.8). In contrast, if nitrate levels are elevated and phosphorous is removed from seawater or lowered to concentrations below 10 mg/m3, gametophyte development will terminate (Table 3.9). Thus good gametophyte development requires both nitrogen and phosphorous, with phosphorous being the critical requirement.
For the growth of young sporelings, on the other hand, nitrogen is the critical requirement. When dissolved nitrogen levels are reduced below 500 mg/m3 during the first 28 days after zoospore collection, gametophytes develop very slowly and sporeling growth is severely retarded (Table 3.9). Adequate nutrition levels are therefore very important during the first month of seedling-rearing in culture tanks.
Phosphorous and nitrogen-based fertilizers should be added to the indoor culture system, the amount and rate of fertilizer application varying at different developmental stages. Nutrients are usually dripped into the seawater during the water-cooling process. The following table gives recommended concentrations:
Concentrations of nutrient elements in the culture tanks should be tested every 3–5 days and any deficiency corrected immediately. Required amounts of fertilizer must be calculated based on optimum concentrations for the entire volume of seawater in the indoor circulation system.
v. Monitoring Stages of Sporeling Development
Careful daily observations of sporelings should be made during all stages of germination, growth and development. In their microscopic stages - as embryospores, gametophytes and embryosporophytes - daily observations of growth should be made under a microscope. In addition to observing the density, size and colour of young sporelings, their tissues and cells should also be examined to check whether sporeling plants are infected with bacteria or other harmful organisms. Curative and/or preventive measures should be taken as soon as diseases are identified. When sporelings become visible to the naked eve, observations should focus primarily on growth rate, external form and colour of the sporelings. Again, diseases or signs of contamination should be found as soon as possible so that curative or preventive measures can be taken as quickly as possible.
Before beginning the sporeling rearing season all components of the culture room, including the piping system and the culture tanks, should be cleaned meticulously.
Cleaning the water Supply Equipment
Settling tanks should be cleaned every 2–5 days, depending on rate of accumulation of solids, by draining them and washing away accumulated precipitates.
The gravel-sand-charcoal filters in the filtration tanks should be cleaned every 7–10 days. This is done by “reverse flushing”, where water is pumped under pressure in the direction opposite to the normal filtration flow. The gravel-sand-charcoal filters in the special filtration tanks that are used for filtering recycled seawater should also be cleaned by reverse flushing every 3–5 days. As a general rule, all tanks holding recycled “indoor” seawater should be cleaned before cleaning the tanks holding newly added “outdoor” seawater.
Cleaning the Sporeling Culture Room
Only designated workers should be allowed to enter the seedling-rearing culture room. Before entering, workers should change their footwear, putting on high rubber boots used only for this purpose. Before entering the culture room workers should step into a tank placed next to the entry-door containing a solution of potassium permanganate (KMnO4). Smoking and spitting should be prohibited in the culture room.
Special care must be taken to keep culture tanks clean. When cleaning, culture tanks should be divided into two groups so that substrates with attached sporelings can be moved temporarily from one group of tanks to the other, allowing the vacant tanks to be cleaned. Vacant tanks should then be scrubbed thoroughly to remove weeds, fallen bits of sporelings and settled detritus. The tanks should be well-rinsed with pressure hoses. The same procedure is then repeated with the second group of tanks.
(b) Spray Water Pressure Method
In an adaptation of the previous method, two workers seated at the ends of a water through hold each substrate curtain between them. Cleaning force is applied not from manual pressure but from water pressure created by a spray hose. A third worker, seated between the other two workers, controls the spray hose, moving its nozzle back and forth so that water is forced through the mesh of the substrate material. The water pressure may be adjusted so that less pressure is used in early stages of sporeling development and more pressure is used in later stages. As before, other workers in the seedling-rearing station carry the substrate curtains from the culture tanks to the cleaning trough and return them to the culture tanks when the spray-cleaning is finished. Though this method of cleaning consumes more seawater, its advantages are that speed and efficiency of the operation are greatly improved and less manual exertion is needed for the actual cleaning procedure.
In autumn months when the natural seawater temperature drops below 20° C, summer sporelings should be transferred from the seedling station to intermediate culture rafts at sea. Growing conditions in natural seawater are much better than in the seedling station. Therefore transfer to intermediate culture should be undertaken as early as possible. The following procedures are required during late stages of sporeling growth in the seedling station in preparation for intermediate culture:
i. Increasing Light Intensity in the Culture Room
In the 3–5 day period immediately preceding transfer of sporelings for intermediate culture, light intensity in the culture room should be gradually increased to equal the light intensity at the sea location.
ii. Increasing Water Flow in the Culture Tanks
In the culture room water circulates at an even rate with few fluctuations. Current flow is relatively slow compared with current flow caused by tides and wave action at sea. In preparation for intermediate culture, water flow in the culture tanks during late stages of sporeling growth should be increased.
Timing of Transfer to Intermediate Culture
Timing of intermediate culture depends on: (i) seawater temperature and (ii) size of young sporelings in the seedling station.
When young sporelings are transferred to intermediate culture before seawater temperature falls below 21° C, the rate of development and survival of sporelings is lowered significantly, with many sporelings becoming detached from the culture ropes. On the other hand, intermediate culture should begin as soon as the seawater temperature falls to 21° C, since early intermediate culture will result in early transplantation which, in turn, means a longer grow-out season that will result in significantly higher output at harvest (see Chapter IV).
Size of Young Sporelings
When transfer of young sporelings is delayed they grow too large in the culture tanks. Overcrowding of sporelings decreases water flow in the culture tanks and lowers illumination levels. These adverse conditions may result in weakened plants, broken blades and increased incidence of diseases. On the other hand, if sporeling plants are too small at time of transfer to intermediate culture, mortality at sea is greatly increased. Optimum size for young sporelings at time of transfer to intermediate culture is 3–5 cm in length.
Transportation of Sporelings to the Raft Culture Site
Finally, care must be taken in transporting sporelings from the seedling-rearing station to the sea site for intermediate culture on raft ropes. Truck transport is used for distances of 100–300 km. When using truck transport, culture mats or other substrates with attached sporelings should be layered in the truck's flatbed, with sporeling plants touching between layers of substrates. No more than 10–12 layers of substrate curtains should be piled on one another, to avoid damage cause by crushing. The layered culture mats should be well-wetted and, on windy days, the load of sporelings should be covered with a tarpaulin to prevent loss of moisture. If the journey is long, additional seawater should be sprayed over the plants at periodic intervals to prevent drying. Transport should usually be done at night when the air temperature is lowest and when traffic conditions are best.
Seedlings attached to substrate mats are loaded in similar fashion when transport is undertaken by ship along the coast. Alternatively, seedling mats may be immersed in large seawater tanks. The seawater should be renewed a few times on route, depending on the length of the journey. Sometimes blocks of ice in plastic bags may be placed in the tanks to lower the seawater temperature, thereby optimizing conditions during transport.