ptifcrand lvticroalgae Culture Systems. Proceedings ofa U,S. Asia Workshop. Honolulu, Hl,199i. !Thc Oceanic Institute

The Developmentof a Phytoplankton ProductionSystem as a SupportBase for FinfishLarval Rearing Research

Vernon Sato The Oceanic Institute Makapuu Point Waimanaio, Hawaii 96825 U.S.A.

ABSTRACT Theuutial focus ofresearch inthe Finfish Program atThe Oceanic Institute wasthe control ofmaturation andspawning intwo species; themiHcfish, Chanos c'hanos, andthe striped mullet Mugil cephalus. Success in thisarea required a reliable livefeeds production systemto support larvalrearing research. Livelarval feeds arecultured intwo stages. Phytoplankton arecultured iathe first stage. Two species havebeen produced and tested,Yannochloropsis ocu1ara Eustigmatophyceae! andTctrascbnis retrarhele Prasinophyceae!. Thealgae arcfcd to the rotifer, Brachionus plic'atilis,in the second stage,While B.plicatilis isthc organism ingested by fishlarvae, thenutritional valueofthis package isstrongly influenced bythe algae thatit is fed. This paper deals withthe development ofa system foralgae culture andthe problems encountered. Progress madein rotifer culturetechniques andlarval nutrition atThe Oceanic Institute isdescribed inTamaru etal. this volume!.

INTRODUCTION opedin bivalvelarviculture in whichdiferent speciesof algaemay be raised as the primary Thecontrol of reproductionis a sig- food for the larvae. In addition,some or- nificantbarrier to progressin fish culture, ganisms,such as penaeid shrimp, may be First,maturation andspawning incaptivity rearedon algaeduring the early larval stages mustbe accomplished. The next major hurdle bu"ust beweaned onto zooplankton in later isthe deve1opment ofsuccessful larvalrearing stages. techniques-The complexity of these techni- The culture of somespecies of finfish quescan vary greatly,depending on thebe- requiresmaintenance of a morecomplex series "aviorand food requirements ofeach stage. of culturesystems. In thesimplest terms. a In thesimplest form of larvalrearing, larvaeare weaned toa driedor formulatedfeed foodpyramid must be established and control- immediatelyfollowing yolk absorption. A led to providethe right foodorganism for each s«ghtlymore complicated system has devel- stageof larvaldevelopment. This may include 258 Sato

cu1turesystems for differentspecies of phyto- foundationfor understandinglarval culture plankton as well as zooplankton. systems.Mr. Eda'scontributions and insights Finfish larval rearing oftendepends on are gratefully acknowledged. the production of phytoplanktonas well as zooplankton. Phytoplanktonserve as feed for the zooplankton which, in turn, nourish the OVERVIEW OF THE FINFISH fish larvae, Different sizes and types of zoo- PROGRAIVI plankton may be raised for different larval stages. Feedsproduction at all stagesmust be The early work done in the Finfish Pro- consistentin qualityand quantity for thedura- gram at The Oceanic Institute dealt with iden- tion of the hatcherycycle if larval rearingis tifying the conditionsunder which milkfish, to be successful. Chanoschanos, could be broughtthrough the Phytoplanktonand zooplanktonproduc- later stages of maturation and induced to tion canoccupy the majorityof the spaceand spawn.Maturation and spawning soon became labor allocated to larval rearing. Phyto- a realityand questions relating to eggviability plankton production generally requires the and larval rearing had to be answered. most space. Therefore, any improvementsin Previouswork with striped mullet and the productionof algaehave the potentialto somepreliminary work with milkfish sug- improveoverall hatchery production. gestedthat live rotifers and brine shrimp, Improvements in production at The Arsemia salina, would be suitable larval feeds. OceanicInstitute's finfish hatcheryhave taken This was supportedby a growing literature placegradually over the pastsix years. The basedon the useof rotifers,brine shrimpand overall goal of the program has been to copepodsas feed for the hrval stagesof many developtechniques for thematuration, spawn- speciesof marinefish. Brine shrimpoffered ing andlarval rearingof thernilkfish, Chanos theadvantage of beingcommercially avaihble chanos,and the striped mullet, Mugil ceph- as cysts which cou1d easily be hatched to alus. Previouslarval rearing efforts indicated nauplii in 24 to 48 hours. Speciesor stages that phytoplanktonand zooplanktonculture requiring a smaller food particle size would systemswould be necessaryin the develop- have to be fed smaller organisms, such as mentof hatcherytechniques. Because of this, rotifers. Rotifer production required the researchin algaeculture hasdeveloped as a productionof algae. support activity within the Finfish Program. Consistencyin the quality andquantity This places limits on the types of research of algaeproduced has resulted in the estab- questions that can be addressed. These limita- lishment of a reliable system for rotifer tions, however, have helped to focus efforts production. This is one of the key factors toward developingtechnologies that are prac- contributing to the successof the Finfish Pro- tical, have immediateapplications and can be gramover the pastsix years. Reliable produc- readily transferredto other sites. tion leve1sfor the hnre of milkfish and striped The finfish hatchery system at'The mulletwere eventuallyestablished. Once con- OceanicInstitute is based, in part, on the sistency in larval rearing was established~ Japanesestyle of larval rearing as practicedby changesin systemdesign, hatchery manage- Mr. Hiroki Eda. His efforts haveprovided a menttechniques and nutritional quality co»d Phytoplankton Production in Hawaii 259

be tested and these factors have been cus- water is provided by municipal sourcesand tomizedfor each species. Advancesin larval seawateris availablefrom two wells, the Sea fish biology have allowed the program to Life Park SLP! well locatedalong the Maka- explore the potential of culturing other puu shoreline, and The OceanicInstitute OI! species,such as the threadfinor "inoi," Poly- well located further inland. The SLP well is daelylus sexfilis, and the mahimahi, Cory- sha1low,approximately 10 rn deep and pro- phaenahippurus. Othernew species are also vides seawater at 32 ppt. The OI well is beingconsidered for futureresearch. approximately 80 m deep and draws water Consistent algal production was not with a salinity of 35 ppt. This water has a achieved overnight. Continuous improve- higher manganeseand iron content than the ments in the facilities and culture techniques SLP well water. have been made since our facility was dedi- cated in 1979. These have led to improved Culture Vessels production. Ne seldom have a production surplus, however, as productionincreases are Indoor cultures are brought up througha usually absorbedby the increasingdemands of series of vessels. Cultures are started in 2-liter larval rearing. flasks Fig. 1!. The secondstage consists of Because algae culture is a part of the 20-liter carboys. Glass carboys have tradi- Finfish Program, the bulk of our efforts have centered on fish reproduction and larval rear- ing. The current systemdoes not necessarily reflect the stateaf-the-art in terms of system designor the latestin scientific technology. It haskept aheadof the demandsof larval rearing by anticipating future needsand incorporating new conceptsto help meetthose needs. Fur- thermore,the basicalgae culture routines have beenkept simple so that the technologycan be easily transferredto other situations.

THE ALGAE CULTURE SYSTEM AND ASSOCIATED PROBLEMS

A number of questionsand problems had to be dealtwith as thealgae culture system evolved Theserevolved around the problem of producinghigh quality algae on a consistent basis.An algaeculture room equipped with ai«onditioning,shelves, fluorescent lights a varietyof glassware,fiberglass cylinders and Figure 1. Two-liter flask culture illuminated by aeration lines was available at the start. Fresh a single fluorescent light. 260 Sato

tionally beenused for this stage;for the past Outdoor cultures are taken through a five years,however, we haveused polyethy- seriesof fiberglasstanks containing 500 liters, lenebags Fig. 2!. This haseliminated clean- 5,000liters Fig. 4! and25,000 hters Fig. 5!. ingand breakage problems. The final indoor Transfersbetween stages and for harvesting stageis the 160-literfiber~s cylinders Fig. are madewith submersiblepumps. 3!.

Figure2. Plasticbag culturesilluminated by fourfluorescent lights.

Figure3. Rowsef cylindercultures. Each row of cylindersisil- luminated by eight fluorescent lights fnot shownl. Phytop I ankton Production in H awaii 26k

Temperature Control a distinct range for optimal growth. Some speciesgrow @sterat higher temperatures,but Air conditioning or someother form of these cultures are much more difficult to temperaturecontrol is importantin the man- manage.They may peaktoo quickly andcrash agementof algaecultures. Many specieshave

Figure 4. Five thousand-liter fiberglass culture tank used for outdoor phytoplankton production.

Figure 5. Sectional fiberglass tanks holding 25,000-liter phyto- plankton cultures; the final stage of outdoor phytoplankton produc- tion. The airline distribution systemis visiblein the first tank. 262 Sato

before they can be used.Higher temperatures reached. When aeration and mixing are in- may also have adverseeffects on the nutrition- creased,increasing the exposure of cells to al quality of the algae. light, more dense cultures are achieved. It is important to managecultures so that Simply increasingthe illumination for carboy they are in the active growth phasewhen they andcylinder cultures has also increased max- are transferred or used as food. At 22 -25'C, imum density, however, after a certain point, cultures will peak in four to five days consis- the amount of heat generatedby additional tently. Becauseof this, inoculation, transfers lighting makessuch modifications impractical. and harvesting can be planned, allowing for Outdoor cultures depend upon ambient continuousproduction. solar irradiation which variesduring the year. Lower temperaturesmay also impactthe In Hawaii, the light:dark photoperiod varies growth of contaminating organisms; the from 1 1hours light'13 hours dark during the growth of someprotozoans and bacteria is winter to 13.5 hours light:10.5 hours dark slowed at low temperatures. This allows the during the summer, Summer cultures ex- algae, which are maintainedin exponential periencemore sunlight and warmer tempera- growth,to reacha harvestabledensity before tures. While the increasein available light contaminants become a problem. helps cultures to grow, the warmer tempera- The temperatureof outdoor culturescan- tures can haveadverse effects on somespecies not be controUed. Thesecultures experience of algae. Specieswith a lower temperature higher temperaturesduring summer periods, optimummay be more prone to crashesduring due to the higher atmospheric temperatures summer months. This is particularly true if and longer day length. In Hawaii, summer cultures are not used immediatelyand haveto temperaturesin outdoor culturesmay regular- be held for a few days after reachingharvest ly climb to 30 C whereaswinter temperatures density. Winter culturesmay be slower in may routinely reach26 C. Experiencehas reachinga usableharvest density due to exces- shown that summer cultures of N. ocidata tend sive cloud cover. During periodsof consecu- to be much more unstable than winter cultures, tive cloudy days, a culture may not grow at The winter season, however, presents other all. Furthermore, heavy rain can dilute cul- problems for culture growth. tures and cause them to overflow the tanks. Cultures which do grow to harvestdensity Ught Energy appear to be much more stablethan summer cultures;they can routinely be held for several Illumination for indoor algae cultures is daysafter reaching harvest density. provided by fluorescentlights. Single tubes provide approximately11,000 lux for flask NUtrients cultures. Carboy and cylinder cultures are equipped with multiple fluorescent tubes The standard nutrient medium used for which provide approximately 14,000 lux of algaeculture is the"Medium-F" presented by radiantenergy. Excessheat from the lights is Guillard andRyther 962!. Thereare a num- minimized by air conditioning. ber of variationsfor this mediumincluding the The indoor cultures are probably light- "F/2 medium" described by McLachlan limited when maximum cell densities are Phytoplankton Production in Hawaii 263

973!. Widespreaduse has led to the com- may not be ideal for the seawateravailable at mercial availability of the F/2 medium. other hatcheries. Experience with freshwaterand marine The current OI Algae Culture Medium algaehas shownthat higher nutrient levels, used for indoor and outdoor culture is based particularly nitrogen and phosphorus,may on Miquel's enrichmentsolution as modified extendthe longevityof a culture. This was by Allen and Nelson 91G! for the culture of suggestedby Bischoff'and Bold 963! in work diatoms. A combination of the high levels of with edaphicalgae cultures. Using a triple nitrogen, phosphorusand iron providedby the nitrogen N! variation of Bold's Basal medium, and the trace metal mixture we have Medium BBM 3N! allows maintainanceof developed has enabled us to consistently some cultures for several months without sub- achieve higher rates of growth in N. oculata culture. and T. tetrathele with the sources of water that Increasing the levels of nitrogen and have been tested. phosphorus in the standard F/2 medium im- Outdoor cultures utilize the same high proved growth in somespecies of algae. Cul- levels of nitrogen and phosphorusas indoors, tures were stableand usablefor longerperiods but cheaper, commercially available agricul- of time, with growth continuingat a decreased tural-grade components are also utilized. rate after the initial period of exponential Theseare generally readily available in other growth. countries, making technology transfer It has recently been observed that the simpler. trace metal solution of the Medium-F may be inadequate under certain conditions. The Filtration and Sterilization specific trace metal component that may be limiting has not yet been identified. Higher Filtration and sterilization of incoming culture densitiescan be attainedby increasing seawateris probablythe most importantmeans the concentration of trace metals in the of preventing contamination in indoor algae medium. The problemappears to be related cultures. There are also various methods of to the source of culture water and may be a removing contaminants from culture media, sitespecific problem. It maybe that modifica- including filtration, autoclaving and hypo- tions should be made to the trace metal for- chlorite disinfection. mulationdepending on thecharacteristics of Stock cultures are maintained in media the available seawater. For this reason, we that has been sterilized via an autoclave, have developedand are currently testing a Stocksare transferredregularly using standard morecomplete trace metal mix. This formula- microbiological techniquesadapted for algae tionis basedon publishedrecipes McLachlan isolation and culture. Guillard 973!, 1973; Pringsheim 1946; Provasoli 1963, Hoshawand Rosowski 973! and Pringsheim 1968!and analysis of commerciallyavailable 946! presenta numberof methodsthat have mixtures.The final formulationshould pro- beenused to isolateand mamtainpure cultures vide a complementof tracemetals that are of algae. In order to minimize exposureto suitabIefor thespecies o falgae cultured at The contaminating organisms, stock cultures are OceanicInstitute. Unfortunately, this mixture maintained separately from the indoor algae culture room, Sato

All water usedin the algaeculture room Species Selection is filtered through 5- and 1-pm cartridge fil- ters. An ultraviolet sterilization system was A number of factors should be considered usedin the past. Thesesystems may lose their when selecting a.species of algae to be cul- effectiveness if not properly monitored and tured. I'n finfish larval rearing,the primary rnamtained. Previousoccupants of the algae concernis that the alga be easily cultured and culture room were not consistent in their use satisfy the nutritional requirementsof the lar- of the ultraviolet sterilizer or the cartridge vae. Because rotifers must be cultured as an filters and there were problems with con- intermediatestage, it is also importantthat the taminants which became established. Current- rotifers grow well on that alga. ly, filters are usedto removelarge particulates Many speciesof algaehave been grown but u1travioletsterilization is not being used. and testedas aquaculturalfeeds. Their varied Carboys and cylinders are filled with sizes and shapesaffect their suitability for seawaterand disinfected with sodium hypo- different organisms. High lipid algaeare able chlorite. After aerating for 24 hours, the to provide for the caloric needs of larvae. residual hypochlorite is neutralized with Furthermore, highly unsaturatedfatty acids, sodium thiosulfate according to the recom- or HUFAs, are consideredto be important in mendations of Hemerick 973!. After another developinglarvae and proteinsare essentialto 24 hours the seawateris readyfor enrichment growth and development. These and many and an inoculum. other factors canbe consideredwhen selecting Outdoor cultures are prepared in a an algal species. similar manner.A 35-pm meshbag filter or a A unicellular eustigmatophyte called 5-pm cartridge filter is usedto remove larger Japanese chlorella," or Nannochloropsis particles from the incoming seawater. The ocuiata, was tested for milkfish and mullet 500- and 5,000-liter tanks are disinfected with larviculture. It was relatively easyto culture sodium hypochlorite and neutralized. This and appeared to outcompetecontaminating minimizes contamination. The 25,000-liter organisms. Rotifers grew well on N. ocular cultures are not chlorinated. This is the final and larvae exhibited survivals of 20 - 30%. culture stage, and errors in chlorination or As a result, the OI larval rearing systemwas dechlorinationcan havedevastating efFects on centered around N. oculata. rotifer production and/or the fish larvae, so Experiments were later conducted to chlorination has been eliminated from this determineif rotifer productioncould be im- stage. If contaminantshave not been a prob- proved by culturing different speciesof algae. lem up to this point, they generally do not Terraselmis tetraiheie, Terraselmis chuii, become one. Chaetocerosgracilis and Isochrysis galbana All culture vessels are cleaned and disin- wereconsidered Because preliminary experi- fected after use. A mild solution of hydro- ments indicated that T. letralheie may have chloric acid is usedto removeany residueson someadvantages over N. oculata, further ex- the sides of the cylinders and tanks. All periments are being conductedwith this vessels are then rinsed well before being species. refilled with seawater. Phytoplankton Production in Hawaii 265

Contaminants as a pasteand stored in a refrigeratorfor later use. The algae may then be usedas feed for Contamination is a major problem in a rotifers or as inocula. number of algae culture systems. Facilities One of the most importantcharacteristics that have adequatelyaddressed this issuehave of species like N. oculata is their ability to beenable to maintainconsistent production for simply outcompetecontaminating algaeand years. For example, the Biological protozoans.Nannochloropsis oculata may not Laboratory,National Marine FisheriesSer- be palatableto grazingprotozoans. It may also vice in Milford, Connecticut has adapted be more effective in competing for nutrients, sterile microbiological techniques for all However, the medium being used is so phasesof their indoorculture system Vkeles nutrient-rich it seemsunlikely that the algae 1973, Wickfors 1990!. Their methods are could effectively tie up all of the excessfor its quiteinvolved, requiring the sterilization of all own use. media and materials used in the inoculation, One alternative explanationis chemical transfer and harvesting of cultures in test interactions,or allelopathy. Somespecies can tubes,fiasks and carboys. Somecu1tures have inhibit the growth of other organisms by beengrowing continuouslyfor over four years releasing toxic" compounds. This type of and yields are comparableto thoseachieved in interaction may explain why cultures of N. bioreactors The Milford systemwould not be oculata remain relatively free of contaminants cost effective on a commercial scale, but it when cultures of other speciesare easilytaken does demonstrate the potential of a well over. managedsystem, Contarninants have not been eliminated Backup Cultures from the system at The Oceanic Institute. Many problems are inherent in the original One of the most important components designand operation of the system. Problems of a good larval rearing run is goodcommun- with contaminants have been minimized, how- ication. Everything depends on food being ever, by preventing them from becoming es- available on a regular basis, Becauserotifer tablished in production cultures. This is production is dependent on phytoplankton accomplished with sterilization and disinfec- production, shortcomings in phytoplankton tion and by maintainingcool temperatures. production can have a significant impact on Culturesare managed as batch cultures and are larval rearing success.Increased demands for movedthrough the systemas quickly as pos- rotifers must be communicated to those sible. In this way, contaminantsrarely be- responsible for rotifer production and the comenumerous enough to cause problems. needsof rotifer production must be communi- Holding cultures beyond the normal catedto thosewho producethe phytoplankton. growth cycle allows contaminantsto become Because indoor phytoplankton production established,so we havedeveloped a protocol must begin sevenweeks before spawning,any for dealingwith suchcultures. Tanks that are changesby the rotifer production team must ready for harvesting but will not be used be anticipated and communicatedas early as immediatelyare concentrated by means of a possible. «ntinuouscentrifuge. The cells are harvested 266 Sato

In theabsence of adequatecommunica- tionand/or foresight, it is importanttohave cylinderstake another seven days to reach backupcultures atall stages.In thisway, harvestdensity. increasingdemands can be addressed almost Theoutdoor routine is basedon a four- immediatelyand adjustments canbe made in orfive-day cycle at each of the three stages. theproduction schedule to supplement those Thisadds another 14 days to the production needs.Backup cultures are routinely main- routine.Rotifers must be brought into the tainedat 30 to 50%in excessof whatis productionroutine one or twoweeks before required.By staying ahead ofthe anticipated thefirst spawn. needsofthe rotifer culture and larval rearing Thegrowth of indoorand outdoor cul- groups,adequate amounts of algaeare almost turesfollows the growth pattern exhibited in always available. Figure6. Firstthere is a lagor acclimation phase,which is followed bythe logarithmic or exponentialgrowth phase. The density or STAGESOF ALGAECULTURE biomassof cells increases at the fastest rate duringthis phase. The duration of the lag phasecan usually beshortened bytransferring Algaeculture at The OceanicInstitute cultureswhile they are still in theexponential takesplace in threestages: stock culture main- growth phase. tenance,indoor culture and outdoor culture. Thetransition phase ischaracterized by Eachstage isdependent onthe earlier stages a declininggrowth rate. This usually leads to toprovide clean cultures ofgood quality. a stationaryphase inwhich no net growth or increasein biomass occurs. Photosynthesis Algae Culture Schedule andcell division may still occur during the All algaeculture activities must be plan- stationaryphase, but the number of newcells nedtwo months before the first anticipated producedwill beapproximately equal to the spawn.Spawning schedules and experimental numberof cellssettling out of the water designsfor larvalrearing trials which relate to columnand/or dying. Therefore, nonotice- algaeproduction must be carefuHy outlined so thatall stages ofproduction canbe properly coordinated. Algaeproduction begins approximately sevenweeks before the first anticipated spawn. Betweenseasons, the algae culture room is cleanedand repairs and modifications com- pleted.Flask cultures started from test tubes takeabout ten days to reacha harvestable density.Each successive stageof culture may takefive or six days before it can progress to thenext. Bringing up cultures from test tubes to carboys/bagstakes about 21 days, and FigureG. Generalized grow!'h pattern of phYto- piankton cultures. Phytoplankton Production in Hawaii 267

able growth occurs. If culturesare held long Stock Culture Maintenance Routine enough, they may enter the death phase or "crash," a rapid decreasein biomass. Each month, two or three tubes of each Cultures at all stagesfollow the same speciesare subcultured;more tubes are pre- growth pattern. All other conditionsbeing pared for speciesin greater demand.Media is equal,the rate of growth andthe final culture autoclaved in the test tubes beforehand,and density at the stationaryphase may vary aliquots from each selected tube are trans- dependingon the size and shapeof the culture ferredto four new tubesusing standard sterile vessel, as well as the amount of aeration or microbiologicaltechniques. Descriptions of mixing. someof thesetechniques modified for algal isolationand culture may be foundin Gui11ard Algae Stack Culture Room 973!, Hoshawand Rosowski 973! and Pringsheim946!. After all of the transfers Stock cultures are maintained in a Stock havebeen completed, the tubesare setin racks Culture Room. This room is located and beneathfluorescent lights. For thenext month, maintainedseparately from the normal culture eachnew culture is invertedonce each day. facility. It is equippedwith an air conditioner Growthin the newculture tubes can usually to maintainrelatively constanttemperatures be observed within a week and is allowed to throughoutthe year. Six shelvesare available continue for a inonth before the next set of for cultures and some small-scaleexperi- transferstakes place. At this time, two or ments. Each shelf is illuminated by a 4-ft. three tubes of each speciesare selectedfor long fluorescenttube. A refrigeratoris used transferring, while test tubes which show no for storing certain chemicalsand autoclaved growth are discarded. Culturesfor starting media. Finally, an incubator is also available production flasks are selected from the for maintainingcultures on a longterm basis. remaining culture tubes. In additionto stockculture maintenance, Seawater for use in stock culture main- the stock culture room is also used for isolation tenance comes from the SLP we11 which work. Interesting algal speciesobserved in providesseawater at 32 ppt. This water is ponds or from natural blooms in the oceanare considered to be abiotic. The standard OI sometimesbrought in for isolation and sub- IndoorA1gae Culture Medium is usedto main- sequentculture. New cultures acquiredfrom tainstock cultures. All of themarine species outside sourcesare usually re-isolatedbefore cultureddo well on this medium. When fresh- they are used. In this way, pure culturesare water species are cultured, Bold's Basal readily availableand unwantedorganisms are Medium3N is used Bischoffand Bold 1963!, excluded from the Stock Culture Room. On and Spirulinacultures are grown with rareoccasions, stock cu1tures may need to be Zarouk'sMedium Zarouk 1966!. re-isolated because of contamination. Temperaturein the Stock Culture Room Although our focus is primarily on waneswith theseason. In general,it fiuctuates marinespecies, some freshwater algae have between20 and 25'C over the courseof a alsobeen isolated and maintained. Liquid and year. Lighting is constant.The incubatoris solid mediacan be preparedaccording to the maintainedat 15 C witha shortday cycle. needsof the project. Sato

By maintaining clean stocks of algae, harvestdensity. The primary flask inoculates contamination problems which have pre- three to six additional 2-liter flasks, the viously impededconsistent, long-term algal "secondaryflask cultures." Thesetransfers productionhave been virtually eliminated. may be staggeredover several days so that Contaminants are recognized early in the harvestableflask cultures are availableevery production cycle and those cultures are day. replaced by new stocks, which are prepared %hen sufficient secondaryflask cultures on a monthlybasis. In addition, stock cultures have been prepared, carboy cultures can be havebeen given to a numberof other research, started. New flask cultures are inoculated commercial and educational facilities. with approximately400 ml from anotherflask culture. The remaining 1400 ml is used to Indoor Cutture System start a carboyor bag culture. The preparation of enough flask cultures for the production Indoor algae culture takesplace in three routine takesabout 21 to 28 days. stages. Culture volumes range from 2-liter Flask cultures reach the highestdensities flasks, to 20-liter carboys/bagsto 160-liter in the indoor culture system. Inoculation is cylinders.Seawater for algaeculture is avail- approximately 10 to 20 million cells/ml and able from the SLP well which hasa salinityof flasks reach a harvest density of 100 to 150 32 ppt. million cells/ml in five to sevendays. The The temperature of the algae culture generalgrowth pattern observedfor flask cul- room is 20 - 25'C. The temperaturevaries tures is presentedin Figure 7A. Maintaining with the season,being higher during the sum- cultures at thesehigher densitiesallows us to mer months. Illumination is constant; inoculate at higher densities and results in provided by 6-fI:. long, high output fluorescent shorter times to reach harvestdensity. After tubes. 21 to 28 days,when enough secondary flask The OI Indoor Algae Culture Medium is cultures have beenprepared, carboy/bag cul- used for all stagesof indoor cultures. This tures can be started. Four hundred milUliters formulation contains high nitrogen and phos- from one flask is used to inoculate another phorus levels and a complete range of trace flask while the remaining 1,400 ml is usedto metals. start a carboyor bag culture.

Indoor Production Routine Carboy/Bag Cultures

The first stageis the preparationof 2-liter Thesecultures were originally housed in flasks. All flask cultures are flied with media glasscarboys which hold 16to 20 liters. Over and autoclavedprior to inoculation, The ini- thepast few years,we havebegun using plastic tial inoculationtakes place in the StockCulture bags,which are sorhewhat easier to handleand Room. The contents of a single test tube are donot requirecleaning. The hazard of broken added to a flask of autoclaved medium. This or chippedglass edges is alsoeliminated. The "primary flask" is taken to the algae culture 5-rnlthick polyethylene bags measure 16 x 24 room and aerated. Flask cultures started from inches.They must be storedand handled to test tubesgenerally take 10 to 14 days to reach avoid puncturingduring the assemblyprocess- Phytoplankton Production in Hawaii 269

The preparation and managementof cultures may be added after the second day. Ap- is the samewhether glass or plastic is used. proximately 1,400 ml of culture is added. The bags are filled with 16 to 18 liters of These cultures will reach harvest density in seawaterthat has beenpassed through 5- and five to sevendays. 1-pmcartridge filters. The waterand bag are Carboy cultures are inoculatedat a den- disinfected with commercial bleach, a 5.25% sityof 7 to 10million ceUs/ml.Harvest density solutioii of sodium hypochlorite, It is heavily is between 40 million and 70 million cells/ml. aeratedovernight and neutralizedwith sodium The typical growth pattern observedin car- thiosulfatethe following day; then the solution boys can be seenin Figure 7B. If all of the is againaerated overnight. Media and algae disinfection steps are properly taken, con- tamination is rarely, if ever observedat this stage. If contaminationis observed,new cul- tures must be brought up immediately to replacecontaminated ones. The bag cultures are usedas inocula for two different phasesof production. In the indoor systemthey are usedto inoculate 160- liter cylinders that are prepared in the same way asthe carboy/bagcultures, The cylinders are cleaned and filled with seawater. Sodium hypochlorite is addedto disinfect the system; after 24 hours the residual hypochlorite is neutralized with sodium thiosulfate. After an additional 24 hours, nutrients may be added, A single bag inoculateseach cylinder. Cylindersare inoculatedat a densityof 5 to 8 million cells/ml. They are usually har- vested at 30 to 60 million cells/ml in seven days. The growthpattern is shownin Figure 7C. This graph is from 1990/1991data and is somewhat atypical of normal cylinder growth. The growth rate was higher than normally observed harvest density was reachedin three to five daysrather than six to sevendays, as previously observed. In addi- tion, manyof the final densitiesexceeded the 60 million cells/ml that is usually expected. Cylinder culturesare usedfor small-scale FIpure7. Growthpattern ofindoor cultures larval rearing and rotifer culture experiments - .. durlnp 1990/799/, AJ Tvvo-literflask culture, as well as in the larval rearing system. They ~-..- @ Tvventy-litercarboy culture, C! 160-/iter cY»nderculture. The harvest densJ'ty ranges also serveas an emergencysource of algaein ereindicated on eachgraph. the rotifer production system

E 270 Sato

Bagcultures are also used to inoculatethe and neutralized with sodium thiosulfate. The outdoor 500-liter tanks. The total indoor seawater is nutrified and the inoculum added. productionperiod from flask to theharvest of The growth performanceof the 500-liter carboys/bagsand/or cylinders takes between tank cultures is presentedin Figure 8A. In- 28 and 35 days. oculationdensity variesbetween 2 million and 5 million cells/ml; the targetharvest density is Outdoor Production System 20 million cells/ml. This generallytakes three to four days during the summermonths. Cul- The outdoor systemconsists of three tures usually take longer, four to five days,to stages.Fiberglass tanks are usedexclusively. reachthe samedensity during winter months. These range in volume from 500 liters to Cloudy weather and rain sometimesprevent 25,000 liters. The seawaterfor outdoor cul- culturesfrom reaching20 million cells/ml. turesmay come from theSLP well, salinity32 The 500-liter cultures are used as the ppt, or from theOI well, which hasa salinity inoculum for the 5,000-liter tanks. Approx- of 35 ppt. imately4,000 liters of seawateris addedto The nutrient medium for outdoor cultures each tank and disinfected with sodium hypo- is based on that used indoors. However, chlorite. This mixture is neutralized with agricultural-grade fertilizers, available through commercialsources, are usedinstead of laboratory-gradereagents, The formula- tion is high in nitrogen and phosphorusand this seemsto promoterapid growth and sustain cultures for several days when they are not used immediately. A complete trace metal mixture is also added.

Outdoor Production Routine

Cultures are started from carboys/bags from the algae culture room. The algae in eachtank is brought up to a inaximumdensity of 20 to 30 million cells/ml beforeproceeding to the next step, Each stagetakes four to five days. Thus,approximately 14 days are neces- sary before the microalgaeis readyto be fed to rotifers. The first stageis the inoculationof up to five 500-liter tanks with culture from the in- Figure8. Growthpattern of 500-litertank cul- door production system. These tanks are tures Al and 5000-liter tank cultures 8/in the outdoor phytop!ankton production system, cleaned and dried beforehand. Seawater is Target harvest density is 20 million cell+'ml. added and disinfected with sodium hypo- Growthpatterns ar e presentedfor summerand chlorite. This mixture is aerated overnight winter seasons Phytoplankton Production in Hawaii 271

sodium thiosulfateafter 24 hours. Following Yearly Growth Patterns in Outdoor neutralization, the nutrients may be added, Production Systems followedby an inoculumfrom a 500-litertank. The target density at harvest for the The summerand winter growth patterns 5,000-liter tanks is 20 million cells/ml.This for the outdoor productionsystem are distinct- is usuallyattained in four to five daysduring ly different. The slowergrowth rateand lower the summer months. During the winter harvest density in the winter are generally period,the cultures may take five to six days attributed to shorter days, lower light inten- to reachthe target density. Also, in the winter sities and lower temperatures. 15 million cells/ml may be the inoculation Light levels are not monitored,however, density. salinity and culture temperature are mon- Most of the four 5,000-liter tanks are itored. A decreasein salinity accompaniedby used during normal production. However, lower temperatureswould be indicative of a additional 5,000-1itertanks may be maintained cloudyday with reasotiablyheavy rainfall. An as backups. These cultures may be used as a increase in salinity and higher temperatures sourcefor new cultures or they may be fed to would suggest a day with high solar irradia- rotifers. tion. The 25,000-liter tanks are used for the The number of days per month of in- third stageof outdoor production. Up to five creasingor decreasingsalinity is presentedin 25,000-liter tanks may be required during the Figure 10. This data is from 1989. Corres- peak of the larval rearing season. The tank ponding temperaturedata is presentedin Fig- walls are scrubbed and disinfected between ure 11A. Heavy rains, increasedcloud cover, cultures. Approximately 20,000 liters of lower temperaturesand lower levels of solar seawater is added to the tarik. Nutrients are irradiation occurred during the months of added,followed by 5,000 liters of inoculum. January and February and early in March of Cultures in the 25,000-liter tanks are 1989. The production data for 1989 is usually inoculatedwith 1 million to 5 million presentedin Figure 118. The valuesrepresent cells/ml. They reachthe target density of 20 the percentage of cultures each month that mioioncells/ml in four to 6vedays during the reached the target density of 20 million summermonths. During the winter, the cul- cells/ml. Less than 30% of the cultures turesmay not reachthe targetdensity until day six. Representativegrowth curves for 25,000- liter culturesare in Figure9. Approximately 6,000 liters of algae is requiredeach day to maintainrotifer cultures, so each tank can be used for three days of feeding,after which theyare drainedand cleanedin preparationfor a newculture. Algae production is staggeredto provide a con- tinuoussupply of algae. Figure 9. Growth pat't'em of 25,000-liter tank cultures. 7arget harvest density is 20 million cell+'m/. Growth patterns are presented for summer and winter seasons. 272 Sato

reached the target density from January creasing demands of rotifer production for through March. larval rearing researchfor the past six years. Late March and April appearto be tran- Production goals have been established at a sition periods to summer conditions. May, minimum densityof 20 million cells/ml for the June, July and August are typical of the sum- eustigmatophyte Nannochloropsis ocuhua. mer periods with high incidentlight levelsand Except for periods of poor weather, these higher temperatures.The larval rearing season goals have been consistently achieved ends in Septemberor October. More than throughoutthe year. The datapresented in this 80% of the cultures reachedthe targetdensity paperis primarily from the 1989larval rearing of 20 million cells/m1during this period. seasonsfor mullet and milkfish. During that The number of outdoor production cul- year, approximately 2 million liters of algae tures is reduced after the end of the larval were producedand the demandfor algaecon- rearing season,between October and Novem- tinued to increase. When spaceis not avail- ber. Cultures are not begunand harvestedon able for increasing the production area, a regularschedule, Temperaturesdo begin to attempts havebeen madeto increaseproduc- decrease in November. While the weather tion by refining techniquesfor managingcul- may become more rainy and cloudy, these tures; thereby improving growth rates and conditions do not present a problem until harvest densities. December and Januarywhen cultures for the There are seasonal differences in the next larval rearing seasonare again brought growth of outdoor cultures. Winter cultures outdoors. appearto be limited by cooler temperatures andless available light. It appearsthat growth

SUMMARY

The phytoplanktonproduction systemof The Oceanic Institute has supported the in-

Figure 10. Number of days with an increase or decrease in salinity of algae cultures. The data is summarired per month for 1989. An in- Figure 11. Ai Average temperature of phyto- crease in salinity suggests warm days with plankton cultures for each monthin 1989. PV high light intensities. A decrease in salinity is Percent of cultures which reach the target caused by rain and suggests cooler weather density of 20 million ceilslml, The datais sum with heavy cloud cover. marized for each monthin 1989. Phytoplankton Production in Hawaii 273

during the winter monthscan be improvedby Handbook of Phycological Methods. Culture Methodsand Growth Measurements.Cambridge increasing light, This can be achieved by University Press, London. pp. 69-85. increasing light intensity or lengthening the Guillard, Robert R.L. and J.H, Ryther. 1962. Studies photoperiod.Providing additional light in an of marine planktonicdiatoms. I. Cyclorellanana Hustedt and Deronula confervacea Cleve! Gran. efficient mannerappears to be the next major Can. J. Microbiol. 8:229-239. hurdlefor both the indoor andoutdoor produc- Hemerick,G. 1973.Mass culture. In: J.R. Stein Ed.!, tion systems, Handbook of PhycologicslMethods. Culture Methodsand Growth Measurements.Cambridge This paper has discussedthe problems UniversityPress, London. pp. 255-266. encounteredby the phytoplanktonproduction Hosbaw, R.W. end J.R. Rosowski. 1973. Methods for team at The Oceanic Institute along with microscopicalgae. In: J.R. Stein Ed.!, Handbook of PhycologicalMethods. CultureMethods and resolutionswhich have beenimplemented. It Growth Measurements.Cambridge University is hopedthat they will providesome insight Press, London. pp. 5348. for thosewho are developingnew production McLachlan,J. 1973. Growth media marine, ln: J,R, Stein Ed.!. Handbookof PhycologicalMethods. systems.The mostserious problems must be Culture Methods and Growth Measurements, addressedfirst. For example,the problem of CambridgeUniversity Press, London. pp. 25-51. light limitation was secondaryuntil problems Pringsheim, E.G. 1946. Pure Cultures of Algae. CambridgeUniversity Press, London, 119 pp, with water quality, nutrients, aeration and Provasoli,L. 1963. Growingmarine seaweeds. Proc. contaminationwere resolved, To optimize Intern. Seaweed Symp. 4:9-17. Pergamon Press, production, eachproblem areamust be clearly New York. Provasoli, L. 1968. Media and prospects for the cul- identified, defined and resolved according to tivationof marinealgae. In: A. Watanabeand A. the local situation. Hattori Eds.!. Culturesand collectionof algae. Proc.U.S. JapanConf. Hakone.Sept. 1966. Jap. Soc. Pl. Physiol.pp. 63-75. Ukeles, R. 1973. Continuousculture a method for LITERATURE CITED the productionof unicellularalgal foods.In: J.R. Stein Ed.!. Handbookof PhycologicalMethods. Allen, E.J. and E.W. Nelson. 1910. On the artificial Culture Methods and Growth Measurements. cultureof marineplankton organisms. J. Mar. BioL Cambridge University Press, London.pp. 233- Assoc. 8.421 254. Bischoff,H.W. andH.C. Bold. 1963.PhycoIogical Wickfors, G. 1990. Personalcommunication. StudiesIV. Somesoil algaefrom Enchanted Rock Zarouk, C. 1966. Contribution a I'Etude d'une andrelated algal species, Univ. TexasPub. No. Cyanophycee.Influence de DiversFacteurs Physi- 6318. Austin,Texas. 95 pp, queset Chinuquessur la Croissanceet al Photo- Guillard,Robert R.L. 1973.Methods for microflag«- synthesede Spirulina maxima. Ph.D. Thesis, latesand nannoplankton. In: J R Stem Ed-! Universityof Paris, Paris. Abstractin French!. 274 Sato Rotifcrand Microalgae Culture Systems, Proceedings of a U.S.- AsiaWorkshop. Honolulu, Hl, l991. ! The oceanicinstitute

Heterotrophic IVlicroalgae Production: Potential for Application to Aquaculture Feeds

Raymond Gladue Martek Corporation 6480 Dobbin Rd. Columbia, Maryland 21045 U.S.A.

ABSTRACT

Phytoplanktoncomprise the base of thefood chain in themarine environment and, as such, are essential toall life in theoceans. Therefore, it is notsurprising that a numberof marineorganisms seem to havean absoluterequirement for microalgae in their diet at somestage in their development. Recently, research has indicatedthat this requirement isdue at least partially to the need for long chain e3 highly unsaturated fattyacids {co3HUFAs! synthesized bythe algae. Due to the importance ofphytoplankton feeds to aquaculture, many producersof bivalvemollusks and crustaceans must devote significant portions of theirtime and resources to growingalgae. In manycases, the algal production systems used are inefficient and expensive. Martek Corporation,a U.S. company devoted tomicroalgal biotechnology, is engaged ina programtodevelop inexpensive,nutritious nucroalgal feeds for aquaculture through theuse of heterotrophic {without light! growth conditions.

BACKGROUNO: HETEROTROPHY derive energy from organic compoundsare VS. PHOTOAUTOTROPHY virtually universal, many photoautotrophic algaehave been found to becapable of hetero- Algae are, for the most part, photo- trophic growth Droop 1974, Neilson and autotrophs, meaningthat they derive their Lewin l974!. Even amongthose algae that energyfrom light and the carbon necessary for displayno heterotrophiccapability, many can buildingbiomass from carbon dioxide by the grow at an increasedrate by simultaneously processof photosynthesis.Heterotrophs, on utilizingorganic carbon and light energy mix- theother hand, acquire both their energy and otrophic growth; Droop 1974!. carbonrequirements from organic compounds Photoautotrophiccultures of algaeuseful ln the form of sugars,fats, organic acids or as feedsfor aquacultureare usually produced any number of other substrates. Since the through one of two methods: metabolicpathways by whichheterotrophs 276 Gia due

AIA Id d limitednumber of locations Kyle and Gladue doorponds is relativelyinexpensive since it 1990, Soong 1980!. relieson naturalsunlight for energy. How- By contrast, industrial fermentationsof ever, this methodis only suitablefor a few, heterotrophicyeast and bacteria often produce fast-growingspecies due to problemswith cultureswith densitiesof 40 140 g/liter. contaminationby predators,parasites and Heterotrophicalgal densities in thisrange have "weed" speciesof algae. Furthermore, beenattained at Martekand at other facilities productionoutdoors requires a largearea of Soong 1980!. At these densities the same suitableland and is subjectto weatherunpre- amount of algae normally produced in a dictability. 10,000liter tank at a hatcherycould be grown Tdi *I d AI I in a bench-top10-liter fermentor. Besides the grower to produce unialgal cultures while obvioussavings in theamount of spaceand maintaining somecontrol over cont:unination. water usedfor algal cultures, there would be However, due to space,energy and skilled a concomitantsavings in thelabor and pump- laborrequirements, it is moreexpensive than ing capacityinvolved in handlinglarge volume outdoor culture. cultures. Heterotrophy has certain advantages Productioncosts for outdoor pondsof over photoautotrophy when it comes to the photosyntheticallygrown algae are in the production of biomass. rangeof ca. US$4- 20/kgdry biomass De- Pauwand Persoone 1988!. However, these Production Costs outdoorculture systems must cope with con- taminationby predatorsand weed species of For photosyntheticalgae, light can be algae,and rely on goodweather and sunlight. thoughtof as an essentialnutrient, without The hidden costs of outdoor culture include whichthe cells cannot grow. Whenlight or poor batch-to-batchconsistency and unpre- any other essentialnutrient! becomes limited dictableculture "crashes" caused by changes in supply,it will slowgrowth and possibly in weather,sunlight and water quality. In affect the biochemicalcontent of the cells. In addition,many algal species that are desirable algalproduction systems, light is almostin- as aquaculture feeds are not suitable for out- variablythe growth-limiting nutrient. This is door culture Rytherand Goldman 1975!. becauseas the algae grow they effectively Productioncosts for indoor photosyn- shadeeach other from the light source. In most thetic algaeculture range from $160to more algal feedproduction systems, either indoors than $200/kg of dry biomass DePauwand or in open ponds,growth becomes light- Persoone1980!. These high costs are partially limited whenthe culture cell densityreaches 6 offsetby theadvantage of beingable to control ca.5 x 10 cells/ml.This density corresponds the algal speciesbeing grown andthe condi- to ca. 100 mg of dry biomassper liter of tions of culture. However, indoor culture culture. Higherdensities of 5 g/liter or more requiresa tremendousamount of spaceand can be attainedby increasingthe light inten- timeand is oftensubject to theculture crashes sity,but the energy cost of thisapproach is too that plagueoutdoor culture. expensivefor indoorculture and is onlyap- Heterotrophic production of microalgae propriateoutdoors for a fewalgal species at a canbe performedfor lessthan $20/kg of dry Heterotrophic Microalgae 277

biomassusing large fermentorsas cultureves- difficult to control DePauw and Persoone sels Soong 1980!. To date,this approachhas 1988!. With the two to three orders of mag- been economically feasible with very few rutudedecrease in volume affordedby hetero- strainsof algae. However, calculationsbased trophic production,as well as the sophisticated on standard models for large scale fermenta- control equipmentthat is startdardwith most tions Kalk and Langlykke 1986! for someof fermentors, quality control can virtuaBy the heterotrophicalgae being testedat Martek eliminate batch to batch variations. projectcosts of $2 - 25/kg. Improvementsin growth rates and process conditions could Nutritional Value lower these production costs to less than $1/kg, as is the case with some bacteria It has beenrepeatedly demonstrated that Cruegerand Crueger 1989!. the nutritional value of algal feedsvaries sig- nificantly betweenspecies and even between Contamination Control different strains of the samespecies Enright et al. 1986, Ryther and Goldman 1975!. This Contamination of algal cultures, either variation can affect growth rates, mortality, by predators such as rotifers, copepodsand time to maturation and disease resistance of protozoa, or by unwanted"weed" speciesof the fish or shellfish consumers see Volkman algae,is a constantthreat with both openpond et al. 1989, for references!.Mixtures of algal and indoor photosynthetic algal production strains usually support better growth than any systems. This often means loss of an entire one strain alone Enright et al. 1986!. Yet, crop, Contamination of a heterotrophic culture due to space limitations and the growth char- by fungi or bacteria can be equally devastat- acteristics of different algae, most aquaculture ing, but the methodsused to prevent such an operations can only afford to supply a few occurrence are more sophisticated. Hetero- different strains for feed. These strains are not trophic cultures are grown in fermentors that always the most nutritious so much as the are specially designedto allow steamsteriliza- easiestor leastexpensive to grow, The reduc- tionof themedium and vessel prior to inocula- tion in cost allowed by heterotrophicproduc- tion. Furthermore,air enteringand leaving the tion should allow more experimentationwith vesselis filter sterilizedto preventtransport of mixtures of different strains as feeds. Even- any microbes. These methodshave proven tually, specific diets promoting optimal effectivein industrial microbiologyover the growth of bivalves and crustaceansat each Past50 years Crueger and Crueger 1989!, growth stagemay be formulated.

Quality Control HETEROTROPHIC PRODUCTION Quality control of photosynthetic algal SYSTElVlS' FERMENTORS feedsdepends on waterquality, control of nutrientsrequired by thealgae, and physical Fermentation technology developed over Parametersthat affect growth such as light, the past five decadesfor the large-scale in- temperatureand pH. In bothindoor tanks and dustrial production of bacteria and yeastscan outdoorponds these factors can be extremely be readily adaptedto heterotrophicalgal 278 G ladue

production.The samefermentor vessels used for other microorganismsare suitablefox growingheterotrophic algae Fig. 1!. Off-the- shelfinstrumentation is availablefor monitor- ing andcontrolling a numberof parameters that affect cell growth andbiochemical con- tent. In addition,a wealthof informationis availablein the industrialmicrobiological literatureon methods for optimizingproduc- tivityand minimizing costs. All thatprevents theroutine fermentative production of micro- algaeis thelack of knowledgeconcerning their heterotrophicpotential.

HETEROTROPHICPOTENTIAL OF IVIICROALGAE Figure 1. Schematicof a standardstirred-tank fermentor,instrumentation for measurement Microalgaeare generally considered to andcontrofis availabie for thefollowing be strictlyphotosynthetic organisms. How- parameters; temperature, pressure, air flow ever, a numberof studieshave demonstrated rate, turbidity, pH, dissolvedoxygen and agita- thatthere are a significantnumber of species tion speed. with heterotrophicor mixotrophicgrowth capability Droop 1974,Hellebust and Lewin 1977,Neilson and Lewin 1974!. Furthermore, trophicallyunder these conditions. However, theseheterotrophic species exist within nearly "obligate"photoautotrophy is only useful to everytaxonomic class of algae Table 1!. describealgal behaviorfor the exact condi- Mostof thestudies that have investigated tionsunder which the tests were performed. heterotrophyin algaehave approached the In manycases, algae labelled as "obligate" questionfrom an ecologicalpoint of view photoautotrophshave subsequently been Droop1974, Hellebust and Lewin 1977!. shownto haveheterotrophic growth ability. Theprimary concerns have been the amount Twoexamples will illustratethis point. of organiccarbon removed from the aquatic 1! Brachiomonassubmarina and some strainsof Haemarococcuspluvialis can reduce environmentby algae compared to bacteria or protozoa!and whether algae can supplement nitratewhen growing photosynthetically but photosynthesisby uptakeof organiccom- areunable to doso in thedark. When supplied with a reducednitrogen source such as am- pounds. In most cases, these studies have testedalgal growth in mediacontaining low monia,however, they are capable of hetero- trophicgrowth Neilsonand Lewin 1974!. concentrationsof organicmatter, as would be foundin the natural environment. Many algae 2! Prymnesiumpanurri and Chroomonas havebeen labelled obligate" photoautotrophs Pyrenomonas!salina are unable to grow het- asa resultof their inabilityto growhetero- erotrophically on the low concentrations of glycerol that might be found in the natural Heterotrophic Microaf gae 279

Table 1. Algal generaused as aquaculturefeeds and/orcapable of heterotrophicgrowth.

Algal class GenUs Used as feed Dark growth Bacillariophyceae Actinocychcs Amphora Bellerochea Coscinodiscus Chaeloceros Cyli ndrotheca Cyclotella Ditylurn Melosira Nitzschia Navicula Phaeodactylwn Skeletonema Thalassi osira

Chlorophyceae Anfastrodesmus Astrephomene Brachiomonas + + Bracteococcus Carteria Chlamydobotrys CKamydomonas Chiorella Chlorococcum Chlorogonium Dunaliella Gonium Haematococcus Neochloris Nannochlons Oocystis Polytoma Polytomella Prototheca Scenedesmus Spongiochloris Stephanosphaera Stichococcas Vohndina

Chrysophyceae Qtromulina Monas Ochromonas Poteri oochromonas 280 Gla due

Table 1. Continued.

Algal dass Genus Used as feed Dark growth Cryptophyceae Chilomonas Chroomonas Cryptomonas Pyrenomonas! Hemi selmis Rhodomonas

Cyanophyceae Anabaena Anacystis Aphanocapsa Calothrir Chlorogloea Fremyella Lyngbya Nostoc Oscillatoria Phormidium Plectonema Spirulina Tolypothrix Westiellopsis Dinophyceae Crypthecodini um Pyrrophyta! Gymnodinium Gyrodinium Gonyaular Heterocapsa Oxyrrhis Pro< ocentrum Scrippsiella Peridinium!

Eugienophyceae Astasia Euglena Eustigmatophyceae Nannochloropsis Marine Chlorella! Prasinophyceae Micromonas Pyramimonas Tetra selmis Heterotrophic Microalgae 281

Table 1. Continued.

informationin this lablc was adapted from the foUowing references; DePauw and Peraoonc 1988, Droop 1974, Guillard 1975, HcllcbLjsrand Lewin 1977, Ncilson and Lcwin 1974, Ukcles l980,

environment,although they can do so when bolism of one substrate will not necessarily suppliedwith very high concentrations.25 inhibit growth on other substrates.Therefore, M! Droop 1974!. one strategy to induce enzyme-deficientalgae There are three main hypothesesexplain- to grow heterotrophicallyis to supply a ing the biochemicalbasis of obligatephoto- mediumcontaining a varietyof carbonsour- autotrophy in algae in experimental settings ces, capableof being catabolizedby different Droop 1974, Neilson and Lewin 1974!. pathways. These hypothesescan also be used to devise 8 f methods to circumvent the biochemical catabolized, a substrate must first be trans- deficienciesresponsible for lackof growthin ported across the cell membrane into the cell. thedark. The postulatedcauses and the strat- Many algae apparently lack efficient uptake egiesused to attack them are listed below. mechanisms for organic carbon sources :S * Ig I k Droop 1974,Hellebust and Lewin 1977!. To theenzymes necessary for thecatabolism of overcome this nutrient transport deficiency certainorganic substrates Droop 1974,Neil- algae can be tested fox heterotrophicgrowth s«and Lewin 1974!.This is generallytrue of on substrates that fit one of the following the blue-greenalgae cyanobacteria!,which criteria: lackone or moreof theenzymes of the tricar- m Substratesthat are widely utilized by boxylicacid cycle and thus cannot grow on algae, as reported in the literature Hel- acetate although at least one blue-green, lebust and Lewin 1977, Neilson and Chlorogloeafritschii, is anexception!. How- Lewin 1974!. ever, thelack of an enzymeinvolved in cata- 282 Gla due

a Substratesthat are capable of somede- gree of passivediffusion acrosscell RESEARCHPROGRAM AT NIARTEK membranes,such as glycerol, acetic acid andlactic acid Neilson and Lewin 1974!. Thealgal feeds research program at Mar- I Substratesfound naturally in thealga's tekseeks to developinexpensive, highly environment.Algae are more likely to nutritiousalgal feeds for aquaculture.This havetransport mechanisms for com- goalis beingapproached from two directions: poundsthat they frequently encounter in I Algaeknown togrow heterotrophically nature.For example, epiphytic diatoms, are being testedas feeds. growingupon seaweed, have been shown to becapable of heterotrophicgrowth on e Algaecurrently used as feeds are being aminoacids and small organic acids ex- testedfor heterotrophic growth potential. cretedby theseaweeds Lewin and Ourapproach for eachalgal strain fol- Lewin1960, Hellebust and Lewin 1977!. lowsa stepwisepattern. : Algae sur- This is vivedaily dark periods by respiring their doneby plating, antibiotic treatment, micro- reservematerials toprovide the energy neces- pipettingor a combinationofaQ three methods saryfor cellmaintenance. However, the ener- Guillardand Keller 1984!. gyproduced during respiration may not be sufficientfor growth or active transport of Screeningis performedin testtube organicsubstrates. In a naturalsetting, this culturesusing media designed tosupport dark characteristicwould be of valueto thecell, growthof fastidiousstrains. These media are Limitedrespiratory capacity would prolong designedon the basisof thehypothetical thesupply of thereserve material, while still causesof "obligate"photoautotrophy asout- providingenough energy for cellmaintenance lined above. untilthe cells were again exposed tolight Neilsonand Lewin 1974!. d~miagd. Thosestrains that exhibit hetero- Algaewith limited respiratory capacity trophicgrowth in thescreen as well as known cansometimes be induced to growhetero- heterotrophsfrom the literature! are grown in trophicallyif they are provided with a high shakeflask cultures todetermine growth rates, energycarbon source as well assufficient nutrientyields grams of biomassper gram of growthfactors, such as vitamins, amino acids substratefor carbon, nitrogen, phosphorus, or otherreduced nitrogen sources! and silicon,vitamins, etc.! and biochemical con- purinesand pyrimidines asbuilding blocks for tents protein, lipid, carbohydrate and~3 fatty nucleicacids. The inclusion of growthfactors acid content!.All of this informationis valu- inthe medium may stretch the limited respir- ablein makingdecisions toscale-up produc- atorycapacity of thealgal cells by eliminating tion of an alga. theneed to synthesize many of these building blocks. sassai. Those algae that exhibit desirable qualities e.g., high growth rate, high ca3 fatty acid content!are culturedin benchscale fer- mentors liters!to determinetheir ability to attainhigh biomass densities 0 g dryweight Heterotrophic Microalgae 283

/liter! and to estimate production costs for leading to this improvementconsisted of large-scaleculture 00 - 200,000liters!. changing the carbon source from sucroseto . Test quan- glucose,instituting pH control, addinga tities of thealgae are produced in fermentors specifictype of' yeast extract and switching - 200 liters!. Biomassis sent to various from a magneticallydriven to a top-driven researchand commercial facilities for testing fermentordesign. At thispoint the alga was as feed for a number of cultured fish and deemedsuitable for scale-upto production shellfish. levels Table 2!. Martek has identified over 80 strains of heterotrophic algae from within its culture collectionof 800+ strains. Testingof these CASE STUDY 2: heterotrophsas feedsis just getting underway. UN DENTIFlED DIATOM Preliminary results indicatethat someof these strainsmay be suitableas feeds for oystersand As part of a continuingscreening pro- rotifers. gramfor microalgalproducers of ~d3HUFAs, diatoms were isolated from seaweed. These diatomswere screened for rapidheterotrophic CASE STUDY 1: growthand for the presenceof significant POTERIOOCHROMONAS quantitiesof thetd3 HUFA, eicosapentaenoic MALHAMENSIS acid EPA!. One of thesestrains was chosen for growth optimizationin our bench-scale Poferioochrornonas formerly Ochro- fermentors. Automatic control of dissolved monas!ma/hamemis is a fiagellatedchryso- oxygen allowed biomass densities to be in- phyceanalga that is unusualin its abilityto creased five-fold and increased the content of grow more rapidly heterotrophicallythan EPA Fig. 2!. Adjustmentsin nutrient feed photosynthetically Droop 1974!. By using ratesand inoculum preparation effected addi- basicprocess optimization techniques thebio- tionalimprovements in productivity Table 3!. massproductivity of this algawas increased by morethan ten-fold. The process changes

Table2. Processdevelopment farP. ma/hamemis production.

productivityisidealized asthe specific growth ratex themaximum densityto indicate themaximum potential forcontinuous Thisconcept isused to estimate production costs for cithcr continuous orbatch culture. 284 Gladue

01SADVANTAGES OF FUTURE POSSIBILIT1ES HETEROTROPH1CALGAL PRODUCTlOM Manyreports have demonstrated the im- portanceof thelong chain ~3 HUFAs, eicosa- Whereasphotosynthetic production of algaeis laborand space intensive, hetero- pentaenoicacid EPA! anddocosahexaenoic trophicproduction iscapital and technology acid DHA!,to thegrowth and maturation of intensive,The cost of a fullyinstrumented marinefish and shellfish Ben-Amotz et al. bench-topfermentor with a twoliter capacity 1987,Langdon and Waldock 1981, Olsen isin the range ofUS$5 - 10,000 while a 10,0001989,other references in Volkman etal. 1989 liter productionfermentor would cost more andDevresse et al. 1990!.Most species of than$1 million Kalk and Langlykke 1986!. commerciallyimportant fish and she11fish In additionto the fermentoritself, other seemto havea limitedability to synthesize facilitiesnecessary for productioninclude a thesefatty acids and must obtain them through steamgenerator, an air compressorand a clean theirdiet Ben-Amotzetal. 1987,Langdon roomfor maintenanceand transfer of axenic andWaldock 1981, Sowizral et al. 1990!. culturesof thealgae. Algae,at thebase of thefood chain, are the primarysource of EPAand DHA in themarine Becauseof theserequirements, it is un- likelythat heterotrophic alga1 production environmentandare essential components in wouldbe feasible for most individual aquacul- thediets of manymarine organisms. Vnfor- turefacilities, Instead, due to the importance tunately,those algae that are the best sources of economiesof scale, existing fermentation of EPAand DHA are not always the easiest facilitiesmay be leased for large production strainsto grow and producers often must seffle runs. foralgal species that do not supply optimum amountsof theseessential fatty acids Ben- Amotzet al. 1987,Ryther and Goldman 1975!. Byselecting heterotrophic algal strains highin EPAor DHA it shouldbe possible to provideinexpensive andnutritious algal feeds for fish and shellfish.Some of thehetero- trophicalgae emerging from the screening programmentioned above have significant levelsof ~3 HUFAs, ranging from 2 -25% of thetotal fatty acid content. If theproduction costsfor thesestrains can be sufficiently reduced,it may even become economically Figure2. Effectof varyingdissolved oxygen feasibleto feedthese algae to otherlive feed concentrationson FPAcontent in an uniden- organisms,such as rotifers and Arremia, to tifiedheterotrophic diatom. Two fermentors increasetheir co3 HUFA content. were run parallel with D.0. controlled at less The strict control that canbe exercised than 70% LowD.O.J or greaterthan 50% High D.O.J of saturation. overheterotrophic cultures using standard fer- mentationtechnology may lead to improved Heterotrophic Microalgae 285

Table 3. Process development for unidentified diatom.

See Table 2,

feedsfor aquaculture.By controllingcertain ~ The potential exists for inducing hetero- chemicaland physicalparameters, growers trophicgrowth in manymore species. should be able to influence the biochemical a Heterotrophicproduction of algalfeeds contentsof thealgal strains. Examples include would be less costly than current theeffect of dissolvedoxygen on EPA content methodsof photosyntheticproduction. described above and the effects of nutrient a Well establishedfermentation technology depletionon fat, protein andcarbohydrate may allow more stringent control of and contents Wikfors 1986!. improvementin the nutritionalquality of Additionally,one of thefactors affecting heterotrophicallyproduced algal feeds. suitability of an algaas food for shellfishis the thickness of its cel1 wall. Certain strains of diatoms e.g., Phaeodacrylumrricornttrum! REFERENCES havebeen shown to bepoor feeds due to their Ben-Amotz, A., R. Fishier and A, Schneller, 1987. indigestibility,despite having high levels of Chemicalcomposition of dietaryspecies of marine ~3 HUFAs Ben-Amotzet al. 1987,Ryther unicellularalgae and roti fers with emphasis on fatty andGoldman 1975!. Studiesat Martekhave acids. Mar. Biol. 95:31-36. Crueger,W. andA. Crueger.1989. Biotechnology: A shownthat the silicon content and, presumab- Textbookof IndustrialMicrobiology, Sinauer As- ly, the thickness of the silicic cell wall of sociates,Inc., Sunderland,Mass., USA. diatomscan be modified by limiting the con- Depauw, N. andG. Persoone.1988. Microalgaefor aquaculture. In: M.A, Borowitzka and L.J. Boro- centrationof dissolvedsilicate in the growth witzka Eds.!, Microalgal Biotechnology.Cam- medium.The ability to controlthe silicon bridgeUniversity Press, Cambridge. pp. 197-221. contentmay improve thesuitability of many Devresse,B M, S. Romdhane,M. Buzzi, J. Rasowa, P. Leger, J. Brown and P. Sorgeloos.1990. Im- diatomsas feeds. provedlarviculture outputs in thegiant &eshwater prawn Machrobrachiumrorenbergii fed a diet of Arremiaenriched with n-3 HUFA andphospho- lipids, World Aquaculture.21:123-125. S UIVlMARY Droop, M.R. 1974. Heterotrophy of carbon. In: W.D,P. Stewart Ed.!. Algal Physiologyand Bio- > A widevariety of algalspecies can grow chemistry Botanical Monographs, Vol. 10!. he»otrophicallyor mixotrophically. Universityof CaliforniaPress, Los Angeles. pp. 530-559. Enright,C.T., G.F. Newkirk, J.S. Craigieand J.D. Castell. 1986.Evaluation of phytophaktmas diets 286 Gla due

for juvenile Ostreaedulis L. J. Exp. Mar. Biol. comparativebiochemistry. Phycologia. 13:227- Ecol. 96: 1-13. 264. Guillard, R.R.L. 1975. Culture of phytoplanktonfor Olsen, Y. 1989. Cultivated microalgae as a sourceof feedingmarine invertebrates, In: W.L. Smithand omega-3latty acids.In: J.E. Jonasson Ed.!. Fish, H.H. Chanley Eds,!, Cultureof MarineInver- Fats and Your Health. Svanoy Foundation, Nor- tebrate Animals. Plenum Press, New York. pp. way. pp. 51-62. 29-60. Ryther, J.H. and J.C. Goldman.1975. Microbes as Guillard, R.R.L. and M,D, Keller, 1984.Culturing food in mariculture. Ann. Rev. Microbiol. 29'.429- Dinoflagel]ates.In: D.L. Spector Ed.!. Dino- 443. flageflates.Academic Press, Inc., NewYork. pp. Soong, P. 1980. Production and development of 391-442. Chioreiirt and Spiruiina in Taiwan. In: G. Shelef Hellebust,J.A. and J. Lewin. 1977. Heterotrophic and C.J. Soeder Eds.!. Algae Biomass. Elsevier nutrition. In: D. Werner Ed,!. The Biology of the /North Holland Press, Amsterdam.pp. 97-113. Diatoms. University of California Press,Berkeley. Sowizral, K.C., G.L. Rumseyand J.E. Kinselia. 1990. pp. 169-197. Effect of dietary alpha-linolenic acid on n-3 fatty Kalk, J.P. and A.F. Lsnglykke, 1986.Cost Estimation acids of rainbow trout lipids. Lipids. 25:246-253. for BiotechnologyProjects. In: A.L, Demainand Ukeles, R. 1980. American experience in the mass N. A. Solomon Eds.!. Manual of IndustrialMicro- culture of micro-algae for feeding larvae of the biology andBiotechnology. American Society for American oyster, Crassostreavirginica. In: G. Microbiology.Washington, D.C. pp. 363-385. Shelef arid C.J. Soeder Eds.!. Algae Biomass. Kyle,D3. andR. Gladue.1990. Microalgae as a source Elsevier/North Holland Press, Amsterdam. pp, of EPA. In: N. Bengtsson Ed.!. New Aspectsof 287-386. Dietary Lipids. Benefits,Hazards, and Use. SIK, Volkmsn, J.K., S.W, Jeffrey, P.D. Nichols, G.L Goteborg, Sweden.pp. 161-169. Rogersand C.D. Garland.1989. Fatty acidand Langdon,C.J. andM.J. Waldock.1981. The effect of lipid compositionof 10 speciesof microalgaeused algaland artificial diets on thegrowth and fatty acid in mariculture. J. Exp. Mar. Biol. EcoL 128:219- compositionof Crassostreagigas spat. J. Mar. 240, Biol. Ass. U.K. 61:431448. Wikfors, G.H. 1986.Altering growth and grosschemi- Lewin, J.C. and R.A. Lewin. 1960. Auxotrophy and cal compositionof two microalgalmolluscan food heterotrophyin marinelittoral diatoms.Can. J. speciesby varyingnitrate and phosphate. Aquacul- Microbiol. 6: 127-137. ture. 59:1-14. Neilson, A.H. and R.A. Lewin. 1974.The uptakeand utilizationof organiccarbon by algae:an essayin Rotifezand hticroalgac Cultuie Systems, Proceedings of a U.S.- AsiaWorkshop. Honolulu, Hl, 1991. !Thc Oceanic lnsiiiuie

The Status of Mass production of Live Feeds In Korean Hatcheries

Mi-Seon Park NationalFisheries Research 5 DevelopmentAgency 65-3 Shirang-ri, Kijang-Up Yangsan-gun,Kyong-Nam 626-900 REPUBLIC OF KOREA

ABSTRACT

Thereare now I hatcheriesunder the controi of theNFRDA and 78 privately owned hatcheries inKorea. Larvicultureisbeing conducted forseveral species offinfish and for abalone, pearl oyster, Japanese tigershrimp, fleshy shrimp, etc. Rotifersare used to feedfinfish larvae and Nannochloropsis ocalata and Chlorella sp. are provided tothe rotifers.Feeds for bivalve larvae include Pavlova lurheri, Jsochrysis galbana, Chaetoceros calcitrans and Chaetocerossimplex, while Terraselmis rerrarhela andSkelerojiema costaium arefed to shrimp larvae. In thecase of inarinefinfish, tanks for fishlarvae, rotifers and Chlorclla culture are present in the approximateratioof I: 2: 6,respectively. Thetype of tank used tomass produce rotifers iscoinmonly a 25-ton concretetank and both zooplankton and phytoplankton areusually cultured according tothe batch culture method.

fNTRODUCTION Studieson theproduction of marinelar- vaein Koreabegan at theNational Fisheries Research& Development Agency NFRDA!. Theestablishment of the aquaticanimal hatcheryat the Yosubranch of theNFRDA pavedthe way for thelarval production of animalssuch as abalone, Haliofis spp., top- »ell, Turbocorrturgs, and tiger puffer, Taki- fugu rubripes.There are now 10hatcheries underthe control of theNFRDA Fig. 1, Table 0 and78 privately owned hatcheries inKorea. Beforethe 1980s, species for whichlar- valproduction was a highpriority were shell- fish suchas abalone,Hajiofis discus and Figure 7.7en hatcheries operatedby the Netion- ai Fisheries Research4 DevelopmentAgency. 288 Park

Table 1. The status of NFRDAhatcheries and larvalproduction statistics for 1990.

Hatcheries Year Scale m ! Production

established Site Floor Species Number of larvae space pukcheju 1973 9,217 1,713 Haliotis discus 500,000 Pagrusmajor 200,000 larvae! 3 x 10 fert. eggs! Oplegnathusfasciatus Turbo cornutus 10,000 Chutnunjin 1979 6,442 1,914 Haliotis discus hanruu 400,000 Pleuronichthyscornutus Arctoscopusjaponica Sebastesschlegeli 10,000 Halocynthia roretzi Strongylocentrotus 10,000 intermedi us

Yoch'on 1980 14,378 2,003 Paralichthysolivaceus 10 fert. eggs! Haliotis discus hannai 300,000 Takifugu rubripes Pagrusmajor 100,000 Acanthopagrusschlegeli 100,000 Portunus trituberculatus 600,000

Yong-il 1981 8,880 1,083 Halioti s discus hantuu 300,000 Halocynthia roretzi Parali chthysoli vaceus 20,000 Anthocidaris 150,000 crassls ina

Koje 1982 14,447 2,954 Hahot>s dkscus hannat 100,000 Penaeusjaponi cus 5,200,000 Tadlfugu rubripes Paralichthys olivaceus 65,000 larvae! 32 x 10 fert. eggs! Halo nthta roretn

Wando 1984 12,973 2,878 Haliotis discus hannai 200,000 Paralichthysolivaceus 20,000 Pagrusmajor 50,000 Acanthopagrusschlegeli Sebastesschlegeli 50,000 Mu il ce halus 30,000 IVlassProduction of Live Feeds in Korea 289

Table 1. Continued.

Hatcheries Year Scale[m~! Production established Site Floor space Species Number of larvae Poryong 1985 38,922 2,709 Haiious discus ~ 100,000 Paralichrhysolivaceus Pagrus major Sebastesschlegeli 50,000 Mugil cephalus 100,000 Penaeusjaponicus Penaeus chinensis 6,350,000 1986 14,877 3,145 Hali ops discus hannai 100,000 Sebastesschlegeli 50,000 Pagrus major Penaeus chinensis 500,000 Portunus trirubercularus 1,800 Namcheju 1988 17,450 2,528 Paralichrhysolivaceus 70 x 10 fert. eggs! 50,000 larvae! Pagrus major 200,000

Haliotis discushannai; arkshell, Anadara STATUS OF MASS CULTURE OF broughronii;pearl oyster, Pinctada fucata; LIVE FEEDS andthe tunicate,Halocynrhia rorerzi. Tech- niquesfor the productionof larval marine Hatcherieshave special facilities for the finfishessuch as bastard halibut, Paralichthys culture of live feeds Table 2!. In the caseof olivaceus;red sea bream, Pagrus major' ,rock marinefinfish, tanks for fish larvae, rotifer bream,Oplegnarhusfasci atus;black porgy, andChlorella culture are present in theap- Acaruhopagrusschlegeli and tiger puffer, Tak- proximateratio of I: 2; 6, respectively.The ifugurubripes; and crustaceanssuch as the amount,timing and types of feedsgiven Acan- Japanesetiger shrimp, Penaeus j aponicus; lhopagrusschlegeli and Pagrus major larvae fleshyshrimp, P. chinensis;and blue crab, Porrunustritubercularus have only been are shownin Tables3 and4, respectively. developedsince 1983. PhytopIankton Culture As theculture of thesespecies has ad- vanced,demand for bettermass culture tech- Pearloysters and arkshells are the species »quesfor microalgaeand zooplankton to feed of bivalves now produced at hatcheries in theearly larval stages has increased. Korea. Increasingamounts of Pavlova lutheri and Chaetoceroscalcirrans are used as feed four partsP, lutheri to every part C. cal- cilrans! Table 5!. F/2 mediumand Provasoli 290 Park

Table 2. Live feeds production at NFRDA hatcheries.

Hatcheries Production Live feeds Capacity of Number of Remarks area production live feeds cul- culture tanks m ! area ture tanks for feeds m ! m ! Pukcheju 1,313.2 270 170 Chumun'in 1,101.5 34.7 Yoch'on 1,907,6 308 100 Phytoplankton 50 Zooplankton 30 Zooplankton Yon -il 680.2

Koje 2,720 50 Phytoplankton 10 Zoo lankton %ando 2,400 250 Phytopiankton Zoo lankton Poryong 2,490 179 6 12 Phytoplankton indoors 2 20 Zooplankton 272 150 outdoors! 70 2 3 Puan 2,517 1245 2 2 3 50 12 Namcheju 1,465 191 26 Phytoplankton 26 Zooplankton 20 Zoo l ankton

Namhae 2,174 331 17 indoors! 33 131 1 160 43 outdoors! 70 150 2 1

ES medium Table 6! are generallyused. The if a great deal is needed,it is sterilized with culture processis outlined in Table 7. UV irradiation. Because the production of live feeds Chlorella sp. is usedprimarily to feedthe should keep pacewith the growth of the target rotifer, Brachionusplicatilis. This is because species, we use serial culture, periodically Chlorella sp. grows rapidly, is easyto culture, upscaling by 15 - 20% as the target species and is euryhaline. Either Complesal or grows. Seawateris sterilized by boiling, but Biwang.medium is used for the intermediate culture of Chlorella sp. in one-ton fiberglass Mass Production of Live Feeds in Korea

Table 3. Amount,timing and speciesof feeds given culturedAcanthopagrus sch!egeli .

R: rotifers,A: brineshrimp, T: short-neckedclam, IVI: mincedmeat

Table 4. Amount, timing and species of feeds given cultured Pagrus major .

8: rotifers,A: brineshrimp, T; short-neckedclam, M; mincedmeat

reinforcedplastic tanks Table8!. Tanksare hatcheries, but 100-ton concrete tanks are filled with filtered seawater,then Chlore1lasp. generallyused. The nutrient medium contains and nutrient mediumare addedand aerationis ammonium sulfate 00 g/ton!, superphos- begun Massculture tanks differ between phate5 g/ton! andurea S g/ton!. 292 Park

Table 5. Productionof Pinctadafucata and Arjadarabroughtonii larvae at NFRDAusing Pavlov@ lutheri and Chaetoceros calcitrans as feed.

The harvest density is 8 - 1G x 10 ZOOPLANKTON CULTURE cells/ml. In the winter, when it is cooler than 8'C, heaters are used occasionally to speed Rotifer Culture growth. The main problem encounteredin the The type of tank used to massproduce mass culture of Chlorella sp. is occasional rotifers in Korea differs in accordance with the rapid declinesin cell number.These "crashes" production method, culture volume, duration are accompaniedby a changein the color of of culture, etc., but 25-ton concrete tanks are the culture medium to yellowish-brown. The commonly used. cause of this phenomenon has not been There are two culture methods Fig. 2!: elucidated, however their occurrence is corre- the total harvesting method batch!, which lated with suddenincreases in temperatureor uses small tanks, and the partial harvesting exceptionally long culture periods under low method semi-continuous!,which employs light conditions causedby continuousrain or large tanks. Recently,the former hasbegun to contaminationwith diatoms!. Water is filtered be usedin conjunctionwith a varietyof tank prior to use with a 3 -5-pin filter. sizes. Due to the consistentpurity of the inocula, it is more efficient than the partial harvesting method and cultures can remain stablefor longperiods of time. Also,planned Mass Production of Live Feeds in Korea 293

Table 6. Composition of media used for productionis possib1ewhen total harvesting phytaplankton culture. method is used. If there is a requirementof 10 tons of rotifers/day, five 10-tontanks are preparedand one tank is harvestedeach day. The advantageof the semi~ntinuous method is that rotifers canbe harvestedwhenever they are needed. The first step in the massproduction of B. plicatilis is to addseawater to anundiluted Chlorella sp. culture. The algal densityis then adjustedto 8 - 10 x 10 cells/ml. Tankslarger than 50 tons are not heated, but cultures in 20-ton tanks are maintained at 25'C with a heater. Strongaeration is provided. The inoculation density is 30 - 50 roti- fers/ml, and after four to five days increases to 100 - 200/ml. At this point, the tank is harvested,A portionis reservedto begina new culture and the remainder is fed to fish. In the absence of Chlorel1a sp., baker' s yeastis fedat therate of 1 -2 g/100rotifers /day. If rotifer densityis low or if the.culture is heavily contaminatedwith protozoa, more yeastis added. Yeast is provided twice daily, in the morning and in the evening. Occasionally there will be three feedings/day, but this is rare. If baker's yeastwhich hasbeen enriched with fat or oil is to be used, 0.25 - 1 g/million rotifers is fed. Harvesting is accomplished with a 58 - 63-pm plankton net and an under- waterpump .25 kw, 0.4 kw! or by gravity.

Artemia

To hatch Anemia eggs, pour seawater into a 500-liter incubatorand add 500 g of dry cysts.If thewater can be maintained at28 C, the eggs will hatch into nauplii in 18-24 hours. Ordinarily,the nauplii are collected and given to larvaeand fingerlings along with 294 Park

Table 7. Culture process for bivalve larval feeds.

Figure 2. Diagram of the techniques used ro culture L-type rotifersin Korea. Ghloreltais added to a density af 1-2x 10 eel/am/. Mass Production of Live Feeds in Korea 295

Table 8. Compositionof mediaused for other typesof feeds. Additionally,some Chlorella culture. hatcheriesenrich their Anemia by secondary culturewith oil and~ yeast,emulsified oil plus ChlorelIa sp., etc. 298