Rotiferand Microalgae Culture Systems, Proceedings of a U.S. Asia Workshop.Honolulu, Hl, i99i. QcThe Oceanic Institute Various Methods for the Culture of the Rotifer, Brachionus plicatilis, in Texas C.R. Arnold and G.J. Holt The University of Texas at Austin Marine Science institute P.O. Box 1267 Port Aransas, Texas 78373 U.S.A. ABSTRACT Various metEodsfor culturingrotifers in bothindoor culture tanks and outdoor culture systems are discussed.The use of algaein combinationwith yeast and emulsified oil is discussed,and advantages of the various culture methods are also included. 1NTRODUCTIO N but the most common are single~lied algae suchas Tefraselmis,Otlorella, and1sochrysis. The rotifer, Brachionusplicatilis, is an Others includebaker's and torula yeast,emul- importantfood organismfor the first feeding sified oil enrichment, and artificial diets. stagesof larval marineanimals around the There are advantagesand disadvantagesto world. It is a cosmopolitaneuryhaline species, eachtype of culture. An algaediet helps thus it is very versatilein marine culture. preservegood water quality in the rotifer Pertinent literature citations are covered in culturesystem, but at theadded expense of Wohlschlaget al, 990!. Therotifer varies in maintainingalgae cultures. Yeast is a simple size dependingon strain and culture condi- andinexpensive source of food,but the nutri- tions, with adult sizeranging from 123to 315 tiona1quality of yeast-fedrotifers is deficient pm in length.This allows strains to becultured for manymarine organisms. To eliminatethis for a specific size Yufera 1982,Snell and problem,enrichment of yeast-fedrotifers has Carrillo 1984!. becomepopular Watanabeet al, 1983a!. Rotifers can be grown in seawaterin a wide rangeof salinities.Our studiesindicate 18ppt is the optimumsalinity for thestrain we METHODS use. The itiitial culture was obtained from the National Marine Fisheries Service Laborator- Stock cultures for inoculant or starter ies in La Jolla., California. Feed types vary, cultures are maintainedin 1 - 2-liter flasks. i 20 Arnold and Holt Theseshould be keptin an areaseparate from 40/mlafter being rinsed three times through a the massculture tanks if possible,to prevent 60-pmfiltering cloth, to rid theculture of most contamination. Stock cultures can be main- contaminants.Beginning on Day 2, yeastis tainedon an algaldiet of Isochrysisgalbana fed at 1.5 g/10 rotifers and emulsified oil at at 24 - 25'C anda lightcycle of 12light:12 3 ml/106 rotifers daily until rotifer densit~ dark. Cultures should be restartedperi- reaches50/ml. Thenadd 1- 1.3g ofyeast/10 odically, at least every month or more, rotifers and 2 - 3 ml of emulsified oil/106 dependingon environmentalfactors. rotifers daily until a density of 100/ml is attained.Then feedyeast at 0.6 - 1.0 g/10 6 Culture Methods rotifers andemu1sified oil at 2 ml/10 rotifers until the density reaches150 - 200/ml, at The following methods havebeen used whichtime harvesting may begin Wohlschlag by the University of Texas Mariculture Pro- et al, 1990! Fig. I!. gram over the past ten years with varying To harvest, drain 20 - 25% of the tank degrees of success. Outdoor culture is limited volume through a 48-pmfiltering cloth to to the warmermonths of the year. Rotifers have been cultured in 1.8-m dia., round,flat-bottomed tanks that hold up to 1,800 liters of water; 140-liter conical tanks;160-liter clear, cylindrical fiberglass reinforced polymer sheet! tanks; 50-liter polyethylenebags; and 1.8-mdia., 3,200-liter round outdoor tanks. Tanks are sterilized beforeuse by additionof 2.5ppm bleach for 12- 24hours, rinsed and cleaned. They are then filled with filtered seawater and the salinity is adjusted with dechlorinated fresh water. Temperature is maintained at 24- 26 C exceptin theoutside tanks. All except the 140-liter conical and outside tanks have continuouslight supplied from light banks with 40-watt florescentbulbs aboveor beside thetanks. The conical tanks have no light other than room ceilinglights, andonly sunlightis used for the outside tanks. Method casingbaker's yeast and ernuI- si fied oil Set up 1,800-liter round tank or conical tanksas describedabove. On Day 1, yeastis fed at 0.6 - 0.8 g/liter, emulsified oil at 1,0 Figure 1. Diagramof rotifer cultureusing ml/10 liters and rotifers are inoculated at 10- baker's yeast and emulsified fish oil. Rotifer Culture in Texas 121 collect rotifers. Refill tanks with filtered sea- water adjustedto a salinityof 18 ppt. Add yeastat0.6-0.8 f 10 rotifers aud emulsified oil at 2 - 3 mV10 rotifers. Repeatdaily until the rotifer population declines. Method vsing a gae Isochrysis gai- bana!, yeast and emvisified oil Set up one 1,800-litertank as described above and inoculate with a 12-liter carboy of Jsochrysisgalbana 32,000 cells/ml! and medium .2 ml/liter of F/2 medium!. Methods for the carboy culture of 1. galbana and F/2 medium formulation are discussedby Treeceand Wohlschlag990!. On the second andthird daysadd 0.1 mVliterof F/2 medium, and when the algaedensity reaches 132,000 cells/ml, inoculatewith l - 10 rotifers/ml. When the concentration of algae decreases, beginadding yeast at 50g/tank and emulsified oilat 1 -2 ml/10liters eachday. Whenrotifer densityreaches 100/ml or more,increase the daily ration of yeastand emulsified oil to 0.7 - 1.0 g/106 rotifers and2-3 ml/106 rotifers, respectively. Harvestingrotifers can begin whenthe densityreaches 200 rotifers/ml see Fig. 2!. Drain 15 - 25% of the tank/day. Repeatuntil the rotifer densitydrops. This culture method should maintain rotifer den- sities at 150 - 200/ml for about 30 days Wohlschlag et al. 1990!. Methods using a/gae as the so/e nvtrient source When using algae, two methodscan be used. The algae can either be cultured sep- aratelyfrom the rotifers or both may be grown in the same container. Aigae and rotiiers cuitUred separately Algae Isochrysisgalbana or Tetraselrnis Figure 2. Diagram of rotifer culture metho using algae, yeast and emulsifiedfish oil. chai<! is cultured in an 1,800-liter tank using 122 Arnold and Hort "fish emulsion", an organic fertilizer available to restart an increasein the daily count. Har- in liquid form Treeceand WohlscMag 1990!. vest as neededon a daily basis,but theculture Rotifers are cultured in separate1,800-liter mustbe harvestedas above if thedaily count tanks. All tanks receive continuous illumina- is thesame for two consecutivedays. tion from overheadlight banksand are we11 aerated.The culture room is keptat 24 + 2'C. Algae and rotifers cuitvred together Algae tanks are filled with seawater Fifty-liter polyethylenebags are used as which hasbeen filtered througha 1-pmfilter. culturecontainers. They are clampedand at- The salinity dependson the speciesof algae tachedto a support frame. IUumination is and the requirementof therotifer strain,but suppliedby 40-watt fluorescentlamps wall generally is in the range of 16 - 30 ppt. The light banks!. Aeration is supplementedwith tankis theninoculated with 100liters of algae C02 approximately50 standardcubic feet per stock30,000 cells/ml!. If this muchalgae is hour!, injected every hour for 20 secondsto not available, lower the volume of seawaterin promotealgae growth, A valve is attachedto tank. It shouldtake three to fourdays to reach the top for aeration and seawateraddition. an algal density of 132,000 cells/ml. If a Bags are filled with filtered seawater, smaller volume is used, double the volume nutrientmedium is added F/2! andalgae is with filtered seawaterand fertilizer daily until inoculatedat 500ceHs/ml. Algae is grownfor a volume of 1,800 liters is reached. This threedays, and then rotifers are inoculatedat culturecan now be usedto feedrotifers by 10/ml. It shouldtake approximately four days draining50 - 60%of thetank volume daily to reachmaximum rotifer density. Densities andrefilling with filteredseawater and adding as high as 400 rotifers/ml have beenachieved fertilizer. This shouldbe donedaily evenif with this method.Rotifers are harvested by algae is not needed. drainingthe entire bag batchculture! and bags The rotifer culture tank is filIed with 900 are discarded after use Trotta 1981, Trotta litersof filteredseawater and 900 liters of algal 1983!. culture water. Rotifers are added, at least 1/ml, moreif available.When algae has been Outside culture consumed and the culture water becomes The culture of rotifers outdoors occurs clear, rotifers maybe harvested.This is ac- betweenMarch and November in south Texas. complishedby draining30 50% of the tank The tanks are covered with a 60% shade cloth volume. The densityshould be 100- 150/ml. during the hottest months of the summer to To maintain this rotifer count, refill the tank inaintain the temperature below 30'C. The with algalculture water and repeat daily until 3,200-liter tanks are filled with unfiltered rotifer population declines Arnold et al. seawaterand provided with aeration; the tank 1976!. is allowedto sit for two to threedays until the A modification of this method is to start algae bloom. It is then inoculated with rotifers with 300 - 400 liters of seawater and add 100 at 10/ml. Sixty gramsof torulayeast is added liters of algae. Inoculate with rotifers at 12 daily,beginning on the third day until harvest 15/ml. Add 100liters of algaedaily until the begins.There is daily monitoringof therotifer rotifer count is constantfor two days. It is population,and once the concentrationreaches necessary to harvest at least 40% of the tank 40/rnl,harvest begins. At thattime, 60 g of Rotifer Culture in Texas 123 yeastis addedtwice