A major cost for marine hatcheries is producing microalgae and live feeds for broodstock, larvae and juveniles. In Australia, the main types of live feed are brine shrimp (Artemia sp.) and rotifers (Brachionus plicatilis) although work is under way on several other varieties, including copepods. Systems for producing large quantities of relatively clean, bacteria-free cultures are costly to establish and often very expensive to run. The University of Tasmania Aquaculture Research Facility in Launceston has developed labour-saving devices and techniques which other hatcheries will certainly find useful.
The research and pilot culture programs at the University of Tasmania's Aquaculture Department require live feeds for marine organisms including prawns, sea horses, green back flounder, clown fish and damsel fish.
In addition to microalgal production facilities, there are two temperature-controlled rooms, one for rotifers and one for Artemia.
The systems are all run by one technician, Alexander Sobolewski, a graduate of the Aquaculture Department.
There are now 19 marine microalgal species in stock, all isolates from the CSIRO at Hobart, including diatoms, golden-browns, greens, blue-greens and a red. Alexander has cloned a local freshwater species for freshwater zooplankton production, as well as a marine filamentous algae which is used as an amphipod stock culture feed.
The algal production line starts from 250ml non-aerated glass flasks through to 3L aerated glass flasks and, if required, 10L aerated carboys (plastic bottles). The water is filtered to 0.2 micron and autoclaved to kill any filter-passing organisms. Water for 200L bags requires 0.2 micron filtration only, while the 1,000L tanks use water filtered to 1 micron which is chlorinated and then dechlorinated. Up to 16 bags and three tanks can be cultured at once.
A 3L flask is enough to inoculate a 200L bag or four carboys. The algal production room is kept at 19 C, although the lights push culture temperatures up to 21 C; the photoperiod is 18 hours of light and 6 hours of dark; 5 per cent carbon dioxide is injected into the aeration lines to maintain pH. The nutrient is Gulliard's F2 with the addition of metasilicate for the diatoms.
Alexander says the 200L bag production is kept in the exponential phase by light and temperature control. "I preheat the filtered water for the bags by holding it in a 1,000L tank with a heating unit so there are no temperature changes when the bags are topped up in the winter months. Most bags will last for six weeks, although the greens remain productive for 12 or more weeks.
"Typical 200L bag production includes Pavlova lutheri, Isochrysis and Tetraselmis suecica. Cell densities for the golden browns can reach around 5-6 x 10^6/ml and for green flagellates up to around 9 x 10^5/ml. I know cultures within industry can reach higher densities, but we are an inland site. We have to bring in seawater by tanker and so it is at a premium - I can only afford two top-ups a week. If I had more water I am sure I could grow higher densities," Alexander explained.
Artemia are used for fish and crustaceans, while rotifers are used only for marine fish culture experiments. Alexander uses nine 80L Nally bins for rotifer production; in conjunction with up to six 500L production tanks, the whole system can produce a harvest of 112-150 x 10^6/ml rotifers per day (20-25% net production per day). The room is maintained at 23 C and continuously lit. Oxygen is added to the air pumped into the culture tanks to keep dissolved oxygen levels above 7 ppm.
"I start a stock culture bin with 40L of Tetraselmis silicica plus 20L of freshwater; this gives a salinity of approximately 20 ppt. To this, I add enough freshwater-rinsed rotifers to give a density of 100/ml - I rinse the rotifers for about three minutes on a 65 um screen to remove as many ciliates as possible. When the culture is clear of feed, I add more salinity-reduced algae plus a little baker's yeast.
"When the bin is full, I split it into another 80L bin with a siphon and continue the process to whatever we need.
"In full production, it takes me around 10 days to get eight or nine Nally bins to an operational density of about 150/ml. This is enough culture medium to inoculate two 500L production tanks at a volume of 300L each and a density of 150 rotifers per ml."
The 500L production tanks are started in a very similar fashion to the stock tanks. Around 200L of algae plus 100L of freshwater is used to give a salinity of 20 ppt, and freshwater rinsed rotifers are added to a density of 150 per ml. At the end of the day a half-ration of yeast is also added as a supplementary feed.
Alexander has simplified his feed rates into two density levels. Rotifers at 150-175/ml use 0.75 g yeast for every 1 x 10^6 rotifers; for densities higher than 175/ml he uses 0.85 per 1 x 10^6 rotifers. Half the ration is given in the morning, the remainder in the afternoon. When rotifer densities reach more than 250/ml, they are culled back to around 200/ml.
Alexander has arranged his culture methods to suit the inland site with its restricted saltwater supply. "I generally start my production tanks at 300L on a Friday to coincide with the algal bag top-ups when the densities are highest. The rotifer culture is fed only yeast on Saturday, Sunday and Monday. On the Tuesday the culture liquid is screened down to 150L using special screens to keep the rotifers in the tank. I then siphon the tank bottom to remove detritus, but I leave the tank sides alone. Algae and reduced salinity (17 ppt) seawater are added until the tanks are topped up to either 400L or 500L, depending on the density of the rotifers, with a minimum density of 150/ml. The salinity of the tanks should now be 20-25 ppt.
"The tanks are next cleaned, fed with algae and water exchanged on the following Friday, then on the next Tuesday and so on for two weeks. I then harvest the whole culture, freshwater rinse it and restart in a pre-prepared 500L production tank. This method allows for a total water exchange once a week."
The tanks sit on short platforms around two sides of the room for easy access and Alexander's stock control system keeps track of culture development.
"I have set up the 500L production tanks to allow for the production of good quality cultures with little suspended solids - this reduces the likelihood of bacterial contamination of the predator cultures. I have examined the hydrodynamics of the tanks and have found that by suspending two airstones 75 mm above the tank floor, the solids and wastes will settle on the bottom of the tank, where they can be readily siphoned out."
He sues this 'hydrodynamic' method in the Artemia ongrowing tanks.
The simplicity of the system means that Alexander needs to spend less than eight hours a week on rotifer production. He uses snap lock fittings on the pipes so they can be quickly dismantled and washed through; his unique liquid distribution system can meter out algae and seawater from the algal unit into the live feeds unit. "When I first started work here, I had to bucket half a tonne of algae twice a week. I am sure my arms reached my kneecaps - never again!"
Algae and water are mixed in a 200L plastic bin and a submersible pump transfers the mixture to the rotifer and Artemia rooms. Taps over each drum control the amount of food flowing into the tank, so he can "set and forget" - overflow is stored in a header tank for later use.
Cross-contamination is always a concern when zooplankton are cultured close together, so each species is kept in a separate room with all equipment - buckets, hoses, screens and so on - clearly identified for each room.
"I don't mind if a few Artemia are in my rotifer culture tanks; they can be screened out. In fact, according to the literature, they are a good way to keep ciliate numbers down. Rotifers in the Artemia tanks are a different proposition - these little blighters increase to such high numbers that they out-compete the Artemia for food, and decrease my production potential."
Alexander is a great believer in close inspection to monitor the health of the culture system. He examines samples each day under a dissecting microscope, paying particular attention to the number of eggs per female, the amount of food in the digestive tracts and how the rotifers are swimming.
"In addition to swimming activity, I look for evidence of suspended particles. Foam on the surface of the cultures is also an indication that something is going wrong."
Fellow technician and marine larval fish specialist Craig Thomas uses the rotifers to feed juvenile (day 5 to day 12) greenback flounder twice a day. He needs reliable supplies and has found Alexander's system very useful. "I can harvest what I need whenever I want it. In the past the cultures were sometimes dirty and often low in numbers; now there is always sufficient stock. It's the same with the Artemia cultures, which I enrich with Frippak and algae."
Research on seahorses at the University created a need for adult Artemia, which led Alexander to develop a reliable and cost-effective ongrowing method.
He grows them in three 250L rectangular plastic bins up to 40 cm deep with a matrix of airtubes about 75 mm above the bottom of the tank. Oxygen is injected into the airlines and aeration is controlled to gently mix the culture. "I aim for a dissolved oxygen level of about 100% saturation and above 6.5 ppm DO2 at 26 C.
The Artemia cysts are first decapsulated and then hatched in 10L plastic cones attached to an air source. The aeration keeps the cysts well mixed and allows hatching within 24 hours in the 26 C room; 24-hour lighting assists in hatching and the growout tanks can be covered to promote better feeding in the post-naupliar stages. "When the Artemia have developed a through-gut (Instar II) they are harvested, washed in fresh water and transferred to the culture tanks at a density of 15-20/ml. These tanks are filled with preheated filtered water and around 60L of Tetraselmis suecica.
"It generally takes about two days for the Artemia to clear the food. Then I split the tank into another and add 40L of algae every two days with yeast to supplement the diet each afternoon. I feed the yeast by watching the colour and turbidity and I recommend approximately 5-6 g per tank, to give a density of 0.5 x 10^6 yeast cells/ml."
"I regularly take Artemia samples and examine their gut contents under the microscope. If the gut is almost full to the anal sphincter then I know they are feeding well. If food particles extend below the sphincter, there is too much feed in the water and the Artemia are feeding without digesting the food."
A semi-continuous culture is established by dropping the water level by algal exchange every two days; a half water exchange is undertaken every seven days. After 10-15 days the Artemia have matured and can begin producing nauplii if the conditions remain favourable with plenty of dissolved oxygen and food. There will be a mixture of adults, nauplii and juveniles in the tanks and more Intar II Artemia are added every few days to maintain a density around 1-2 adults per ml.
"The cultures can stay viable for up to 4 weeks, depending on how they are managed."
Alexander uses a series of screens from 150 um to 1,000 um (1 mm) to harvest different size classes of the Artemia from the three tanks in only 20 minutes. "Juvenile marine fish generally can only use 2-3 day old Artemia (less than 500 um). In the past, Artemia that had grown larger than this size had to be discarded; now we ongrow it and use it for a variety of purposes, including seahorses, marine tropical aquarium fish and predator size selection experiments."
For more information contact Alexander Sobolewski, Aquaculture Department, University of Tasmania, PO Box 1214, Launceston, Tas 7250. Tel: 003 243-801, fax: 003 243-804.
(excerpts from article by Dos O'Sullivan in Austasia Aquaculture, Vol.10, No 4, Sept./Oct.'96
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