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Production of Nannochloris spec. (Chlorophyceae) in large-scale outdoor tanks and its use as a food organism in marine aquaculture
Aquaculture, 23 (1981) 171-181 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
PRODUCTION OF NANNOCHLORIS SPEC. (CHLOROPHYCEAE) LARGE-SCALE OUTDOOR TANKS AND ITS USE AS A FOOD ORGANISM IN MARINE AQUACULTURE
U. WITT*,
P.H. KOSKE**,
D. KUHLMANN**,
177
IN
J. LENZ* and W. NELLEN*
*Institut fiir Meereskunde an der Universitlt Kiel, Aussenstelle Biilk, 2301 DiinischenhagenBiilk (Federal Republic of Germany) **GKSS Forschungszentrum GmbH, 2054 Geesthacht-Tesperhude (Federal Republic of Germany) (Accepted
24 June 1980)
ABSTRACT Witt, U., Koske, P.H., Kuhlmann, D., Lenz, J. and Nellen, W., 1981. Production of Nannochloris spec. (Chlorophyceae) in large-scale outdoor tanks and its use as a food organism in marine aquaculture. Aquaculture, 23: 171-181. Difficulties in transferring monocultures of marine microalgae from small-scale laboratory to large-scale open air conditions were overcome by the introduction of the small coccal green alga Nannochloris spec. which was found to be dominant in highly eutrophic waters of low salinity as well as in sea water/waste water mixtures. This species proved to be eurythermic, euryhaline and insensitive to changing nutrient and light conditions, the maximum growth rate lying between salinities of 10 and 200/,, . In aerated outdoor tanks under the experimental conditions, Nannochloris was never overgrown, though, during the course of the year, sometimes contaminated by other algae. Despite its small size (2-6 pm cell diameter), it proved a suitable food item for secondary producers such as rotifers (Brachionus), copepods (Eurytemora), and oysters (Crassostrea). Its adaptability, rapid growth rate and high nutritional value qualify Nannochloris as an optimal primary producer in marine aquaculture enterprises.
INTRODUCTION
A major problem in marine aquaculture is the production of suitable microalgae in quantities sufficient to serve as live food for the second link in the food chain (Goldman and Ryther, 1976; Persoone and Claus, 1978). In 1977 we were also faced with this problem, when we started our experimental work at Biilk at the mouth of Kiel Fjord with the object of investigating the productivity and feasibility of marine food chains in large-scale cultures under outdoor conditions. Preliminary experiments to select those of the commonly known ‘food algae’ (Monbchrysis, Isochrysis, Tetraselmis, Dunaliella, Amphidium) best adapted to open-air conditions showed that, unless the medium is previously
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filtered, all monocultures soon became overgrown with other species. We therefore tried to find an alga already optimally adapted to open-air conditions in respect of salinity, temperature and light, and at the same time suitable as a food item. In the natural mixed populations of the highly eutrophic Schlei Fjord and in cultures fertilized with secondarily treated waste water, a small (2-6 pm diameter) coccal chlorophyte, Nannochloris spec., was found to be a dominant organism. We observed mass cultures of this species for over 2 years in aerated 4 and 27 m* outdoor tanks. No succession took place during this period. The cultures were sometimes contaminated by the diatoms Skeletonema costatum and especially Phaeodactylum tricornutum which, however, did not become dominant. Laboratory and field experiments were conducted to gain information on the physiological, ecological and general suitability of this species as a food item in order to create the best possible growth conditions for mass culture with regard to type and dosage of fertilizers, so as to obtain a maximal yield. METHODS
Nannochloris cultures were obtained from mixed cultures from the Schlei Fjord by serial dilution. In no case were sterile cultures used. The nutrients were biologically clarified waste water from the sewage plant of the Kiel municipality and the agricultural fertilizer ‘Nitrophoska blau spezial@ ‘, containing N in the form of NH4’ and N03-, POa3- (N:P at/at = ll:l), Fe Si and other trace elements. This fertilizer possesses the one drawback of not being fully soluble in water. However, a fully soluble fertilizer of similar composition would be far more expensive. Measurements Cells were counted with a Thoma haemocytometer. Protein was determined by the method of Lowry et al. (1951); NH,‘-N, N03--N, inorganic dissolved PO4 and total phosphorus as described by Grasshoff (1976); seston by the method of Lenz (1971); and chlorophyll a by that of UNESCO (1966). PAR (photosynthetic active radiation) was measured by continuous recording with a quantum sensor Ll-1905 in conjunction with a Lambda Instruments printing integrator Ll-550. Laboratory
experiments
In addition to the fertilizers mentioned above, tests were conducted with pure nutrients. Seawater and waste water were filtered through 0.45 pm membrane filters. Light sources were Philips fluorescent tubes TL 40 W, the light intensity was 9-15 W/m* and temperature was 20°C. In flow cultures, the nutrients were added with a peristaltic pump.
173
Outdoor
experiments
The outdoor tanks were situated on the premises of the municipal sewage plant near Kiel. The tanks were of two sizes, 4 m2 and 27 m2, with a water depth of 25-50 cm. For turbulence and COZ input, aeration was accomplished by means of a Siemens cycloblower. Preliminary experiments were conducted to find the most suitable aeration system that ensured a sufficient input of CO, together with a high turbulence (Bartels, 1978; SchrBder, 1978) Perforated tubes running along the tank bottom were compared with airstones and air-lifts as used by Sorgeloos et al. (1977). Algae production was significantly lower in both the latter aeration systems, especially with air-lifts, which are also impractical for use in large tanks. For our purpose, perforated tubes with holes of 0.5 mm diameter proved to be the least complicated and most economical system. Seawater (15-20 o/oo salinity) for the cultures was directly supplied from the Baltic Sea by means of a pipe line. It was filtered through a 1.5 m thick bed of gravel overlying the intake pipe and pumped into two storage tanks, from where it flowed by gravity into the culture basins. Heating pipes running along the bottom of the tanks, through which warm (15°C) waste water was pumped, provided a heating system during winter. Thus even at outside temperatures well below zero, the temperature in the tanks never dropped below 4-6”C. RESULTS
Growth conditions Experiments with varying salinities (O-30 ‘loo ) have shown Nannochloris to be a decidedly euryhaline organism (Fig. 1). It grows within the entire salinity range tested, the growth maximum lying at a salinity between 10 and it is may be seen from the fact that, given sufficient 20 O/o0. How eurythermic sunlight, we observed doubling times of 2-3 days (cell concentration 2-3 lo6 /ml) even at temperatures of 1°C. In experiments dealing with diurnal rhythms (1ight:dark hours 14:10), the cells grew from 2-3 pm to 4-6 pm diameter during daylight hours; during the dark phase, cells adapted to this rhythm showed a nearly synchronous division rate. The cells divided into two and also four. Parallel to the cell count increase, chlorophyll a production also took place during the dark hours. For this reason, the cell volume was very variable, a circumstance that makes calculating the biomass on the basis of cell counts difficult, as shown in Table I. An exact measurement is not possible during routine counts because of the very small size of the organisms. From a fairly large number of routine measurements and by comparing cell counts, seston, protein and chlorophyll a we arrived at an average diameter of 3.45 pm during the morning hours. We used this value for calculating
,
lo51
I
1
I 2
I 3
I
4
I 5
I 6
I 7 days
Fig. 1. Growth of NannochZoris spec. at different salinities; temperature 20” C.
TABLE I Relation between cell diameter and dry weight in Nannochloris spec. (Calculation assumes dry weight to be 25% of fresh weight, specific weight to be 1, and cell shape spherical) Diameter (ccm)
3.0 3.5 4.0 5.0 6.0
Volume (pm3)
14.1 22.4 33.5 65.4 113.0
Conversion from cell count to weight (dry matter) ( lo6 cells/mg)
(pg/106 cells)
283.7 178.6 119.4 61.2 35.4
3.5 5.6 8.4 16.3 28.2
175
production on the basis of daily measurements of cell concentrations. In laboratory cultures, cell volume also depends on nutrient concentration and light. During the exponential growth phase, the average contents for carbohydrate, protein and lipids were ll%, 42% and 15% respectively (Queisser, 1978). Several studies with fertilizers have shown that when offered NOJ--N and NH4’-N together, Nunnochloris prefers the NH4’ component. This was also observed for other species and seems to be usual with the majority of phytoplankton organisms (Bienfang, 1975; McCarthy et al., 1977; Eppley et al., 1979). Even excessively high nutrient concentrations such as 12 mg N/l and 2.4 mg P/l did not lead to growth inhibition. A comparison between filtered waste water (which contained only NH4’ as N source) and Nitrophoska showed no significant difference in growth and chlorophyll a content as long as the culture medium contained enough NH4’. In cultures where the fertilizer used contained NOJ- as the sole N component, the chlorophyll a as well as the protein content in the cells was observed to decrease (Table II). Since the measurement of dry matter as seston is not ideal (C/N analysis would be preferable), the values for dry matter should be considered only as guidelines. TABLE
II
Effect of various Ncontaining fertilizers on growth and composition of cells. showing laboratory and outdoor cultures (average values for morning measurements)
N% NC, NH, + NO, Outdoors NH, + NO,
P consumption Wglmg dry matter)
between
Chlor. D (% in dry matter)
Protein (% in dry matter)
P (% in dry matter)
N consumption Wglmg dry matter)
12 49 60
0.87 0.42 1.06
31 20 29
0.85 0.77 0.96
51 32 49
8.0 7.9 9.7
16.0 8.9 13.5
185
1.60
32
76
16.8
10.0
Cell. cont. (lo6 /mg dry matter) Laboratory
differences
N/P (at/at)
The fertilizer requirement is calculated on the basis of cell counts. To find the exact doses of N and P required for the production of a certain biomass in terms of cell concentration, experiments were conducted with the aim of avoiding waste of fertilizer when harvesting cultures. In outdoor cultures, the average dry weight yield of algae for an uptake of 1 mg N was 13-20 mg and for 1 mg P 60-125 mg. Comparable data are reported by De Pauw et al. (1978a) for Scenedesmus. The higher values indicate a limitation of the corresponding nutrient. The daily harvest of algae as food for the secondary producers is carried out as follows: when cell concentration has reached the required density of 8-20 million cells per ml, part of the crop is harvested, the amount varying with weather and season (sunlight), and ranging from 25-30s in summer to zero in winter, when a regular harvest is not possible because of light limitation. The quantity of the crop removed daily is calculated in such a way that
176
when the original volume is restored after harvesting, the former cell density should be reached by the following day. This pattern can be repeated, using the required amount of fertilizer as calculated according to the expected increase, until algal growth is inhibited by grazers. The proliferation of grazers makes it necessary to clean the tanks every 2-4 weeks and start new cultures. Therefore spare tanks are required in order to maintain the food supply. In none of the trials was an attempt made to stabilize the pH by blowing free CO? into the cultures in addition to aeration. Thus pH sometimes increased to 10.5. Production capacity The average yearly production calculated on the basis of cell count increase ranged from approximately 14 g dry matter m-* d-’ in June (max. 25 g m-* d-l ) to 1 g dry matter m-* d-l in January. On days with low light (PAR < 2.0 E m-* d-l ), p ro d UCt ion was nearly zero, or even below, due to respiratory losses and sedimentation, independent of water temperature. Dependence of production on temperature was only observed at higher light values. Thus in our latitudes, production of algae without artificial light is only worthwhile from April to October. Light (PAR) conversion efficiency during spring and summer amounted on average to 2.58% (n = 21; max. 5.50%, min. 0.81%), assuming a caloric content of 3.85 kcal/g dry weight for Chlorophyceae (Cummins and Wuyceck, 1971). Food u tilization The algae have so far been fed to rotifers (Brachionus plicatilis), copepods (Fury ternora affinis), molluscs (Crassostrea gigas) and together with zooplankton to mysids (Neomysis integer). Mass cultures of Brachionus plicatilis at densities of approximately 150 ind./ml were reared without difficulty for several months on Nannochloris both in the laboratory and outdoors. An example of the productivity of Brachionus cultures fed with Nannochloris is given in Table III. Experiments were carried out in open tanks. Tank I (1.4 m3 ) was aerated by a perforated tube at the bottom, tank II (2.2 m3 ) had no aeration. The mean temperature was 17.4 f 3.2”C. No efforts were made to adjust pH to the optimum of about 7.5-8.0 for Brachionus cultures (Hirayama and Ogawa, 1972; Furukawa and Hidaka, 1973). During the observation time, average pH was 9.2. Average growth rates were comparable in the two tanks, but the average harvest was greater in the aerated tank, due to higher culture densities. After harvesting, the volume was replaced with algae (mean cont. 16 X lo6 cells/ml). Ingestion rate measurements and direct observation suggests that the amount of algae fed per day was not enough to achieve optimal growth, assuming a maximum ingestion rate of 120 cells min-’ per individual (our own measure-
177 TABLE III Harvest and growth rate of Bmchionus plicatilis fed with Nannochloris in two outdoor tanks; tank I (1.4 ms) aerated, tank II (2.2 m3) without aeration. Harvested volume was replaced with Nannochloris (mean cow. 16 X lo6 cells/ml) Date
23.5 24.5 25.5 27.5 28.5 29.5 30.5 31.5 1.6 3.6 4.6 5.6 6.6 7.6 8.6 11.6 13.6 15.6 18.6 5 27(11)
Cont. before harvest (ind./ml)
Amount harvested (lo6 ind.)
Volume harvested (So)
Growth rate lnNt-lnN, (r = -) t (days) -
Tank I
Tank I
(Tank II)
Tank I
Tank I
(Tank II)
108.4 88.8 90.7 93.2 107.1 36.5 83.2 37.8 170.1 56.7 76.9 39.3 138.6 -
-
46 46 46 46 46 23 46 23 69 35 46 23 46 -
-
-
172 141 144 183 171 116 132 122 180 120 122 125 219 120 91 351 90 214 306 days
158
(Tank II)
(55) (70) (97) (75) (56) (71) (57) (100) (87) (98) (70) -
(79)
-
(34.6) (44.1) (61.1) (94.1)
85.1 220.5 28.4 132.4 189.0
(44.7) (35.8) (62.9) (54.8) (123.5) -
66.0
(50.5)
69 46 23 46 46 28.6
(Tank II)
(28.6) (28.6) (28.6) (57.2) (28.6) (28.6) (28.6) (28.6) (57.2) -
(28.6)
0.44 0.64 0.32 0.78 0.25 0.39 0.55 0.64 0.39 0.44 0.67 0.83 a.12 -0.12 0.46 a.37 0.62 0.35 0.37
(0.58) (0.67) (0.07) (0.25) (0.25) (0.12) (0.55) (0.21) (0.46) _
(0.35)
ment at 16°C) or about 200 Chlorellu cells min-’ per individual at 22°C (Hirayama and Ogawa, 1972). In addition to the algal biomass pumped into the tanks, about 50% might be produced there. The walls and bottom of tank I were overgrown with Enteromorpha, producing sufficient detritus which Bruchionus could graze on. Copepods (Eury temoru affinis) were cultured on Nunnochloris in outdoor tanks of 1.5 m3 and 27 m3 volume. Besides Nannochloris there were always various flagellates, ciliates and detritus in the tanks serving as additional food for the copepods. Therefore it was not possible to get exact data for conversion efficiency. Maximum densities observed were 3000 adults + copepodids and 5000 nauplii per litre. During July and August 1979, a 27 m3 tank with an average concentration of 1000 adults + copepodids and 1500 nauplii was harvested by removing 10% of the volume per day and replacing it by the same volume of Nunnochloris culture. No decrease in culture density was observed. In 1978 and 1979 we obtained Crussostreu gigus spat from Scotland. Some hundred (1978) and 6000 (1979) juvenile oysters were cultured in a 2 m3 tank which was supplied with an air-lift circulator after Salser and Mock (1973), producing a water flow through the trays. Nunnochloris was added daily to obtain a concentration of approximately lo5 cells/ml in the tank.
178
The growth rates observed (Table IV) are comparable to those published by Walne (1972), Epifanio and Mootz (1976), Malouf and Breese (1977) and Briggs (1978), part of which are from laboratory studies, however. The oysters did not grow fast enough to fit into the scheme for a 2-year cycle of production proposed for the German coasts by Meixner (1979). After an initially promising growth for about 3 months, shell development later declined. The reason may possibly be sought not in the food but in the water quality and its relatively low salinity in summer. The 1979 experiment had to be terminated in July because of difficulties with seawater supply. Mortality during the observation period was 14%. TABLE IV Growth of Crassostrea gigas spat fed with Nannochloris in outdoor tanks. Data from two supplementary experiments in 1978 and 1979 (n = 100, monthly means for temperature and salinity, maximum length in brackets) Date
Temperature (“C)
Salinity (“/WI)
Fresh weight (9)
Shell length (mm)
13. 6.79 13. 7.79 13. 7.78 13. 8.78 13. 9.78 13.10.78 13.11.78
16.3 14.5 16.0 17.6 13.5 13.0 13.0
16.0 16.5 17.6 16.3 19.2 20.4 20.5
0.04 0.17 0.27 0.66 0.94 1.30 1.57
6.8 ‘12.3 14.6 18.1 18.8 21.0 22.3
(11) (17) (22) (27) (30) (32) (36)
DISCUSSION
The results of the experiments so far conducted appear to indicate that Nannochloris is a suitable food item for mass cultures. Its advantages over other organisms tested lie in its excellent adaptability to environmental conditions, as evidenced by its dominance in outdoor cultures throughout the year. The temporary contamination by Phaeoductylum and, to a lesser extent, Skeletonemu is caused by temperature changes. The same phenomenon is described by Goldman and Ryther (1976), Goldman (1977) and De Pauw et al. (1978b), for other phytoplankton mass cultures. An additional factor, which may favour the predominance of Nannochloris in our case, is the comparatively low salinity (average 15 o/oo) of the Baltic Sea water used for the cultures. We did not pay special attention to the concentration of silicates as a species determining factor. The agricultural fertilizer used contains 0.71% SiOZ by weight and the content of the sewage water is of the order of 30 mg/l. The sea water pumped to the culture site is filtered through a gravel bed, where it probably takes up silicates. We therefore do not think that silicon deficiency by preventing diatoms from becoming dominant, could be an important factor in our cultures.
179
The production and yield per m* during the year is comparable to other values reported for our latitude (Soeder, 1976; De Pauw et al., 1978b). We are aware of the harmful effects of the high pH values in our cultures which cause a growth inhibition due to lack of CO2 and bicarbonate. Other nutrients are lost by transformation of NH4+ into NH3 gas and precipitation of phosphate. Production could possibly be enhanced by blowing CO2 into the cultures, but this is merely an economic problem. Of particular relevance are the positive results obtained from feeding experiments with Brachionus and Eurytemora, both of which play an important role in our turbot larvae rearing project (Kuhlmann et al., 1981). Since we worked without pre-treated water, thus avoiding the high costs involved, our type of mass culture has the disadvantage of being susceptible to contamination by grazers, especially Brachionus, the ciliate Euplotes and various heterotrophic flagellates. A mass development of these grazers can destroy a culture within a few days. To avoid this, it is advisable to harvest the algae crop in time. In the case of small culture volumes, treatment with approximately 20 ppm of 40% formalin is fairly effective against ciliates and heterotrophic flagellates (Rothbard, 1975). For practical reasons we decided to work with semi-continuous cultures, harvesting not continuously but at intervals. Continuous cultures (chemostat or turbidostat) are superior in theory to these as far as production capacity is concerned, and can also be maintained in outdoor cultures of greater volume (Shelef et al., 1969; Ryther et al., 1975; De Pauw et al., 197813). In connection with feeding exact doses of different volumes from one algal tank to secondary producers in several tanks, it seems impractical to run a continuous flow culture. Despite this, it might be possible to run a combination of, say, an oyster culture together with an algal tank, suitable in volume to the consumers, as continuous cultures. The most economic fertilizer would be purified waste water (Goldman, 1976; Oswald et al., 1977). However, the quality fluctuates depending on the efficiency of the waste water plant. Moreover, there is again the danger of contamination by grazers. Toxic substances may be present and harm sensitive organisms. The more thoroughly the waste water is treated, the greater would be the quantity required on account of the lower nutrient content, the result being an undesirable dilution of the seawater. ACKNOWLEDGEMENTS
We are grateful to Dr. Koch, Pflanzenphysiologisches Institut der Universit& Gattingen for identifying the species and to Dipl.-Biol. Neudecker, Bundesforschungsanstalt fiir Fischerei, Hamburg, for providing the oyster spat. The study was financed by the Federal Ministry for Research and Technology (Project ‘Marine Bioproduction’ ME 0320/7).
180 REFERENCES Bartels, D., 1978. Untersuchung verschiedener Druckluftsysteme zur Turbulenzerzeugung in Algenzuchtbecken. Studienabschlussarbeit in der Fachrichtung Bioingenieurwesen, Fachhochschule Hamburg, 67 pp. Bienfang, P.K., 1975. Steady state analysis of nitrate-ammonium assimilation by phytoplankton. Limnol. Oceanogr., 20: 402-411. Briggs, R.P., 1978. Aspects of oyster culture in Strangford Lough, Northern Ireland. Aquaculture, 15: 307-318. Cummins, K.W. and Wuyceck, J.C., 1971. Caloric equivalents for investigations in ecological energetics. Mitt. Int. Ver. Limnol., 18: l-158. De Pauw, N., Bruggeman, E. and Persoone, G., 1978a. Research on tertiary treatment of swine manure by mass culturing of algae. Mitt. Int. Ver. Limnol., 21: 490-506. De Pauw, N., Verlet, H. and De Leenheer Jr., L., 1978b. Heated and unheated outdoor cultures of marine algae with animal manure. Presented at the First International Symposium on Production and Use of Microalgae Biomass, Acco, Israel, Sept. 1978. Epifanio, C.E. and Mootz, C.A., 1976. Growth of oysters in a recirculating maricultural system. Proc. Nat. Shellfish. Assoc., 66: 32-37. Eppley, R.W., Renger, E.H., Harrison, W.G. and Cullen, J.J., 1979. Ammonium distribution in southern California coastal waters and its role in the growth of phytoplankton. Limnol. Oceanogr., 24: 495-509. Furukawa, J. and Hidaka, K., 1973. Technical problems encountered in the mass culture of rotifer using marine yeast as food organisms. Bull. Plankton Sot. Japan, 20: 61-69. Goldman, J.C., 1976. Phytoplankton response to waste-water nutrient enrichment in continuous culture. J. Exp. Mar. Biol. Ecol., 23: 31-43. Goldman, J.C., 1977. Biomass production in mass cultures of marine phytoplankton at varying temperatures. J. Exp. Mar. Biol. Ecol., 27: 161-169. Goldman, J.C. and Ryther, J.H., 1976. Temperature-influenced species-competition in mass cultures of marine phytoplankton. Biotechnol. Bioeng., 18: 1125-1144. Grasshoff, K., 1976. Methods of Seawater Analysis. Chemie-Verlag, Weinheim-New York, 317 pp. Hirayama, K. and Ogawa, S., 1972. Fundamental studies on physiology of rotifer for its mass culture. I. Filter feeding of rotifer. Bull. Jap. Sot. Sci. Fish., 38: 1207-1214. Kuhlmann, D., Quantz, G. and Witt, U., 1981. Rearing of turbot larvae (Scophthalmus muximus L.) on cultured food organisms and post-metamorphosis growth on natural and artificial food. Aquaculture, 23: 183-196. Lenz, J., 1971. Zur Methode der Sestonbestimmung. Kieler Meeresforsch., 27: 180-193. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the folin-phenol reagent. J. Biol. Chem., 193: 265-275. McCarthy, J.J., Taylor, W.R. and Taft, J.L., 1977. Nitrogenous nutrition of the plankton in the Chesapeake Bay. 1. Nutrient availability and phytoplankton preference. Limnol. Oceangr., 22: 996-1011. Malouf, R.E. and Breese, W.P., 1977. Seasonal changes in the effects of temperature and water flow rate on the growth of juvenile Pacific oysters, Crassostrea gigas, Aquaculture, 12: l-13. Meixner, R., 1979. Culture of Pacific oysters (Crassostrea gigas) in containers in German coastal waters. In: T.V.R. Pillay and A. Dill (Editors), Advances in Aquaculture. Fishing News Book Ltd., Famham, Surrey, England, pp. 338-339. Oswald, W.J., Benemann, J.R. and Koopman, B.L., 1977. Production of biomass from fresh water aquatic systems using microalgae. Proc. Fuels from Biomass Symp., Univ. Illinois, Champaign, IL, pp. 59-81. Persoone, G. and Claus, C., 1978. Mass culturing of marine algae - a bottle-neck in the nursery culturing of molluscs. Presented at the First International Symposium on Production and Use of Microalgae Biomass, Acco, Israel, Sept. 1978.
181 Queisser, C., 1978. Untersuchungen iiber die Wachstumsrate und biochemische Zusammen setzung mariner Mikroalgen bei Abwasserzugabe als Diingemittel. Staatsexamensarbeit, Univ. Kiel, 88 pp. Rothbard, S., 1975. Control of Euplotes spec. by formalin in growth tanks of Chlorello spec. used as growth medium for the rotifer Brachonius plicatilis which serves as feed for hatchlings, Bamidgeh, 27: 100-109. Ryther, J.H., Goldman, J.C., Gifford, C.E., Huguenin, J.E., Wing, A.S., Clarner, J.P. Williams, L.D. and Lapointe, B.E., 1975. Physical models of integrated waste recycling marine polyculture systems. Aquaculture, 5: 163-177. Salser, B.R. and Mock, C.R., 1973. An air-lift circulator for algal culture tanks. In: J.W. Avault Jr. (Editor), Proceedings of the 4th Annual Workshop of the World Mariculture Society, Louisiana State Univ., Division of Continuing Education, Baton Rouge, LA, pp. 295-297. SchrSder, S., 1978. Untersuchung des Algenwachstum bei Verwendung verschiedener Druckluftsysteme zur Turbulenzerzeugung. Studienabschlussarbeit in der Fachrichtung Bioingenieurwesen, Fachhochschule Hamburg, 77 pp. Shelef, G., Oswald, W.J. and Golueke, C.G., 1969. The continuous culture of algal biomass on wastes. In: I. Malek, K. Beram, Z. Fencl, V. Munk, J. &i&a and H. Smrfkovl (Editors), Continuous Cultivation of Microorganisms. Academic Press, New York and London, pp. 601-629. Soeder, C.J., 1976. The technical production of microalgae and its prospects in marine aquaculture. In: K. Tiews (Editor), Fisheries Resources and their Management in Southeast Asia. German Foundation for International Development (DSE), Berlin and Federal Res. Bd. Fisheries, FAO, Rome, pp. 321-333. Sorgeloos, P., Persoone, G., De Winter, F., Bossuyt, E. and De Pauw, N., 1977. Air-water pumps as cheap and convenient tools for high density culturing of microscopic algae. Proceedings of the 8th Annual Workshop of the World Mariculture Society, San Jose (Costa Rica), January 9-13, 1977, pp. 173-183. UNESCO, 1966. Determination of Photosynthetic Pigments in Sea Water. Monographs on Oceanographic Methodology I. UNESCO, Paris, 66 pp. Walne, P.R., 1972. The influence of current speed, body size and water temperature on the filtration rate of five species of bivalves. J. Mar. Biol. Assoc. U.K., 52: 345-374.
PRODUCTION OF NANNOCHLORIS SPEC. (CHLOROPHYCEAE) LARGE-SCALE OUTDOOR TANKS AND ITS USE AS A FOOD ORGANISM IN MARINE AQUACULTURE
U. WITT*,
P.H. KOSKE**,
D. KUHLMANN**,
177
IN
J. LENZ* and W. NELLEN*
*Institut fiir Meereskunde an der Universitlt Kiel, Aussenstelle Biilk, 2301 DiinischenhagenBiilk (Federal Republic of Germany) **GKSS Forschungszentrum GmbH, 2054 Geesthacht-Tesperhude (Federal Republic of Germany) (Accepted
24 June 1980)
ABSTRACT Witt, U., Koske, P.H., Kuhlmann, D., Lenz, J. and Nellen, W., 1981. Production of Nannochloris spec. (Chlorophyceae) in large-scale outdoor tanks and its use as a food organism in marine aquaculture. Aquaculture, 23: 171-181. Difficulties in transferring monocultures of marine microalgae from small-scale laboratory to large-scale open air conditions were overcome by the introduction of the small coccal green alga Nannochloris spec. which was found to be dominant in highly eutrophic waters of low salinity as well as in sea water/waste water mixtures. This species proved to be eurythermic, euryhaline and insensitive to changing nutrient and light conditions, the maximum growth rate lying between salinities of 10 and 200/,, . In aerated outdoor tanks under the experimental conditions, Nannochloris was never overgrown, though, during the course of the year, sometimes contaminated by other algae. Despite its small size (2-6 pm cell diameter), it proved a suitable food item for secondary producers such as rotifers (Brachionus), copepods (Eurytemora), and oysters (Crassostrea). Its adaptability, rapid growth rate and high nutritional value qualify Nannochloris as an optimal primary producer in marine aquaculture enterprises.
INTRODUCTION
A major problem in marine aquaculture is the production of suitable microalgae in quantities sufficient to serve as live food for the second link in the food chain (Goldman and Ryther, 1976; Persoone and Claus, 1978). In 1977 we were also faced with this problem, when we started our experimental work at Biilk at the mouth of Kiel Fjord with the object of investigating the productivity and feasibility of marine food chains in large-scale cultures under outdoor conditions. Preliminary experiments to select those of the commonly known ‘food algae’ (Monbchrysis, Isochrysis, Tetraselmis, Dunaliella, Amphidium) best adapted to open-air conditions showed that, unless the medium is previously
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filtered, all monocultures soon became overgrown with other species. We therefore tried to find an alga already optimally adapted to open-air conditions in respect of salinity, temperature and light, and at the same time suitable as a food item. In the natural mixed populations of the highly eutrophic Schlei Fjord and in cultures fertilized with secondarily treated waste water, a small (2-6 pm diameter) coccal chlorophyte, Nannochloris spec., was found to be a dominant organism. We observed mass cultures of this species for over 2 years in aerated 4 and 27 m* outdoor tanks. No succession took place during this period. The cultures were sometimes contaminated by the diatoms Skeletonema costatum and especially Phaeodactylum tricornutum which, however, did not become dominant. Laboratory and field experiments were conducted to gain information on the physiological, ecological and general suitability of this species as a food item in order to create the best possible growth conditions for mass culture with regard to type and dosage of fertilizers, so as to obtain a maximal yield. METHODS
Nannochloris cultures were obtained from mixed cultures from the Schlei Fjord by serial dilution. In no case were sterile cultures used. The nutrients were biologically clarified waste water from the sewage plant of the Kiel municipality and the agricultural fertilizer ‘Nitrophoska blau spezial@ ‘, containing N in the form of NH4’ and N03-, POa3- (N:P at/at = ll:l), Fe Si and other trace elements. This fertilizer possesses the one drawback of not being fully soluble in water. However, a fully soluble fertilizer of similar composition would be far more expensive. Measurements Cells were counted with a Thoma haemocytometer. Protein was determined by the method of Lowry et al. (1951); NH,‘-N, N03--N, inorganic dissolved PO4 and total phosphorus as described by Grasshoff (1976); seston by the method of Lenz (1971); and chlorophyll a by that of UNESCO (1966). PAR (photosynthetic active radiation) was measured by continuous recording with a quantum sensor Ll-1905 in conjunction with a Lambda Instruments printing integrator Ll-550. Laboratory
experiments
In addition to the fertilizers mentioned above, tests were conducted with pure nutrients. Seawater and waste water were filtered through 0.45 pm membrane filters. Light sources were Philips fluorescent tubes TL 40 W, the light intensity was 9-15 W/m* and temperature was 20°C. In flow cultures, the nutrients were added with a peristaltic pump.
173
Outdoor
experiments
The outdoor tanks were situated on the premises of the municipal sewage plant near Kiel. The tanks were of two sizes, 4 m2 and 27 m2, with a water depth of 25-50 cm. For turbulence and COZ input, aeration was accomplished by means of a Siemens cycloblower. Preliminary experiments were conducted to find the most suitable aeration system that ensured a sufficient input of CO, together with a high turbulence (Bartels, 1978; SchrBder, 1978) Perforated tubes running along the tank bottom were compared with airstones and air-lifts as used by Sorgeloos et al. (1977). Algae production was significantly lower in both the latter aeration systems, especially with air-lifts, which are also impractical for use in large tanks. For our purpose, perforated tubes with holes of 0.5 mm diameter proved to be the least complicated and most economical system. Seawater (15-20 o/oo salinity) for the cultures was directly supplied from the Baltic Sea by means of a pipe line. It was filtered through a 1.5 m thick bed of gravel overlying the intake pipe and pumped into two storage tanks, from where it flowed by gravity into the culture basins. Heating pipes running along the bottom of the tanks, through which warm (15°C) waste water was pumped, provided a heating system during winter. Thus even at outside temperatures well below zero, the temperature in the tanks never dropped below 4-6”C. RESULTS
Growth conditions Experiments with varying salinities (O-30 ‘loo ) have shown Nannochloris to be a decidedly euryhaline organism (Fig. 1). It grows within the entire salinity range tested, the growth maximum lying at a salinity between 10 and it is may be seen from the fact that, given sufficient 20 O/o0. How eurythermic sunlight, we observed doubling times of 2-3 days (cell concentration 2-3 lo6 /ml) even at temperatures of 1°C. In experiments dealing with diurnal rhythms (1ight:dark hours 14:10), the cells grew from 2-3 pm to 4-6 pm diameter during daylight hours; during the dark phase, cells adapted to this rhythm showed a nearly synchronous division rate. The cells divided into two and also four. Parallel to the cell count increase, chlorophyll a production also took place during the dark hours. For this reason, the cell volume was very variable, a circumstance that makes calculating the biomass on the basis of cell counts difficult, as shown in Table I. An exact measurement is not possible during routine counts because of the very small size of the organisms. From a fairly large number of routine measurements and by comparing cell counts, seston, protein and chlorophyll a we arrived at an average diameter of 3.45 pm during the morning hours. We used this value for calculating
,
lo51
I
1
I 2
I 3
I
4
I 5
I 6
I 7 days
Fig. 1. Growth of NannochZoris spec. at different salinities; temperature 20” C.
TABLE I Relation between cell diameter and dry weight in Nannochloris spec. (Calculation assumes dry weight to be 25% of fresh weight, specific weight to be 1, and cell shape spherical) Diameter (ccm)
3.0 3.5 4.0 5.0 6.0
Volume (pm3)
14.1 22.4 33.5 65.4 113.0
Conversion from cell count to weight (dry matter) ( lo6 cells/mg)
(pg/106 cells)
283.7 178.6 119.4 61.2 35.4
3.5 5.6 8.4 16.3 28.2
175
production on the basis of daily measurements of cell concentrations. In laboratory cultures, cell volume also depends on nutrient concentration and light. During the exponential growth phase, the average contents for carbohydrate, protein and lipids were ll%, 42% and 15% respectively (Queisser, 1978). Several studies with fertilizers have shown that when offered NOJ--N and NH4’-N together, Nunnochloris prefers the NH4’ component. This was also observed for other species and seems to be usual with the majority of phytoplankton organisms (Bienfang, 1975; McCarthy et al., 1977; Eppley et al., 1979). Even excessively high nutrient concentrations such as 12 mg N/l and 2.4 mg P/l did not lead to growth inhibition. A comparison between filtered waste water (which contained only NH4’ as N source) and Nitrophoska showed no significant difference in growth and chlorophyll a content as long as the culture medium contained enough NH4’. In cultures where the fertilizer used contained NOJ- as the sole N component, the chlorophyll a as well as the protein content in the cells was observed to decrease (Table II). Since the measurement of dry matter as seston is not ideal (C/N analysis would be preferable), the values for dry matter should be considered only as guidelines. TABLE
II
Effect of various Ncontaining fertilizers on growth and composition of cells. showing laboratory and outdoor cultures (average values for morning measurements)
N% NC, NH, + NO, Outdoors NH, + NO,
P consumption Wglmg dry matter)
between
Chlor. D (% in dry matter)
Protein (% in dry matter)
P (% in dry matter)
N consumption Wglmg dry matter)
12 49 60
0.87 0.42 1.06
31 20 29
0.85 0.77 0.96
51 32 49
8.0 7.9 9.7
16.0 8.9 13.5
185
1.60
32
76
16.8
10.0
Cell. cont. (lo6 /mg dry matter) Laboratory
differences
N/P (at/at)
The fertilizer requirement is calculated on the basis of cell counts. To find the exact doses of N and P required for the production of a certain biomass in terms of cell concentration, experiments were conducted with the aim of avoiding waste of fertilizer when harvesting cultures. In outdoor cultures, the average dry weight yield of algae for an uptake of 1 mg N was 13-20 mg and for 1 mg P 60-125 mg. Comparable data are reported by De Pauw et al. (1978a) for Scenedesmus. The higher values indicate a limitation of the corresponding nutrient. The daily harvest of algae as food for the secondary producers is carried out as follows: when cell concentration has reached the required density of 8-20 million cells per ml, part of the crop is harvested, the amount varying with weather and season (sunlight), and ranging from 25-30s in summer to zero in winter, when a regular harvest is not possible because of light limitation. The quantity of the crop removed daily is calculated in such a way that
176
when the original volume is restored after harvesting, the former cell density should be reached by the following day. This pattern can be repeated, using the required amount of fertilizer as calculated according to the expected increase, until algal growth is inhibited by grazers. The proliferation of grazers makes it necessary to clean the tanks every 2-4 weeks and start new cultures. Therefore spare tanks are required in order to maintain the food supply. In none of the trials was an attempt made to stabilize the pH by blowing free CO? into the cultures in addition to aeration. Thus pH sometimes increased to 10.5. Production capacity The average yearly production calculated on the basis of cell count increase ranged from approximately 14 g dry matter m-* d-’ in June (max. 25 g m-* d-l ) to 1 g dry matter m-* d-l in January. On days with low light (PAR < 2.0 E m-* d-l ), p ro d UCt ion was nearly zero, or even below, due to respiratory losses and sedimentation, independent of water temperature. Dependence of production on temperature was only observed at higher light values. Thus in our latitudes, production of algae without artificial light is only worthwhile from April to October. Light (PAR) conversion efficiency during spring and summer amounted on average to 2.58% (n = 21; max. 5.50%, min. 0.81%), assuming a caloric content of 3.85 kcal/g dry weight for Chlorophyceae (Cummins and Wuyceck, 1971). Food u tilization The algae have so far been fed to rotifers (Brachionus plicatilis), copepods (Fury ternora affinis), molluscs (Crassostrea gigas) and together with zooplankton to mysids (Neomysis integer). Mass cultures of Brachionus plicatilis at densities of approximately 150 ind./ml were reared without difficulty for several months on Nannochloris both in the laboratory and outdoors. An example of the productivity of Brachionus cultures fed with Nannochloris is given in Table III. Experiments were carried out in open tanks. Tank I (1.4 m3 ) was aerated by a perforated tube at the bottom, tank II (2.2 m3 ) had no aeration. The mean temperature was 17.4 f 3.2”C. No efforts were made to adjust pH to the optimum of about 7.5-8.0 for Brachionus cultures (Hirayama and Ogawa, 1972; Furukawa and Hidaka, 1973). During the observation time, average pH was 9.2. Average growth rates were comparable in the two tanks, but the average harvest was greater in the aerated tank, due to higher culture densities. After harvesting, the volume was replaced with algae (mean cont. 16 X lo6 cells/ml). Ingestion rate measurements and direct observation suggests that the amount of algae fed per day was not enough to achieve optimal growth, assuming a maximum ingestion rate of 120 cells min-’ per individual (our own measure-
177 TABLE III Harvest and growth rate of Bmchionus plicatilis fed with Nannochloris in two outdoor tanks; tank I (1.4 ms) aerated, tank II (2.2 m3) without aeration. Harvested volume was replaced with Nannochloris (mean cow. 16 X lo6 cells/ml) Date
23.5 24.5 25.5 27.5 28.5 29.5 30.5 31.5 1.6 3.6 4.6 5.6 6.6 7.6 8.6 11.6 13.6 15.6 18.6 5 27(11)
Cont. before harvest (ind./ml)
Amount harvested (lo6 ind.)
Volume harvested (So)
Growth rate lnNt-lnN, (r = -) t (days) -
Tank I
Tank I
(Tank II)
Tank I
Tank I
(Tank II)
108.4 88.8 90.7 93.2 107.1 36.5 83.2 37.8 170.1 56.7 76.9 39.3 138.6 -
-
46 46 46 46 46 23 46 23 69 35 46 23 46 -
-
-
172 141 144 183 171 116 132 122 180 120 122 125 219 120 91 351 90 214 306 days
158
(Tank II)
(55) (70) (97) (75) (56) (71) (57) (100) (87) (98) (70) -
(79)
-
(34.6) (44.1) (61.1) (94.1)
85.1 220.5 28.4 132.4 189.0
(44.7) (35.8) (62.9) (54.8) (123.5) -
66.0
(50.5)
69 46 23 46 46 28.6
(Tank II)
(28.6) (28.6) (28.6) (57.2) (28.6) (28.6) (28.6) (28.6) (57.2) -
(28.6)
0.44 0.64 0.32 0.78 0.25 0.39 0.55 0.64 0.39 0.44 0.67 0.83 a.12 -0.12 0.46 a.37 0.62 0.35 0.37
(0.58) (0.67) (0.07) (0.25) (0.25) (0.12) (0.55) (0.21) (0.46) _
(0.35)
ment at 16°C) or about 200 Chlorellu cells min-’ per individual at 22°C (Hirayama and Ogawa, 1972). In addition to the algal biomass pumped into the tanks, about 50% might be produced there. The walls and bottom of tank I were overgrown with Enteromorpha, producing sufficient detritus which Bruchionus could graze on. Copepods (Eury temoru affinis) were cultured on Nunnochloris in outdoor tanks of 1.5 m3 and 27 m3 volume. Besides Nannochloris there were always various flagellates, ciliates and detritus in the tanks serving as additional food for the copepods. Therefore it was not possible to get exact data for conversion efficiency. Maximum densities observed were 3000 adults + copepodids and 5000 nauplii per litre. During July and August 1979, a 27 m3 tank with an average concentration of 1000 adults + copepodids and 1500 nauplii was harvested by removing 10% of the volume per day and replacing it by the same volume of Nunnochloris culture. No decrease in culture density was observed. In 1978 and 1979 we obtained Crussostreu gigus spat from Scotland. Some hundred (1978) and 6000 (1979) juvenile oysters were cultured in a 2 m3 tank which was supplied with an air-lift circulator after Salser and Mock (1973), producing a water flow through the trays. Nunnochloris was added daily to obtain a concentration of approximately lo5 cells/ml in the tank.
178
The growth rates observed (Table IV) are comparable to those published by Walne (1972), Epifanio and Mootz (1976), Malouf and Breese (1977) and Briggs (1978), part of which are from laboratory studies, however. The oysters did not grow fast enough to fit into the scheme for a 2-year cycle of production proposed for the German coasts by Meixner (1979). After an initially promising growth for about 3 months, shell development later declined. The reason may possibly be sought not in the food but in the water quality and its relatively low salinity in summer. The 1979 experiment had to be terminated in July because of difficulties with seawater supply. Mortality during the observation period was 14%. TABLE IV Growth of Crassostrea gigas spat fed with Nannochloris in outdoor tanks. Data from two supplementary experiments in 1978 and 1979 (n = 100, monthly means for temperature and salinity, maximum length in brackets) Date
Temperature (“C)
Salinity (“/WI)
Fresh weight (9)
Shell length (mm)
13. 6.79 13. 7.79 13. 7.78 13. 8.78 13. 9.78 13.10.78 13.11.78
16.3 14.5 16.0 17.6 13.5 13.0 13.0
16.0 16.5 17.6 16.3 19.2 20.4 20.5
0.04 0.17 0.27 0.66 0.94 1.30 1.57
6.8 ‘12.3 14.6 18.1 18.8 21.0 22.3
(11) (17) (22) (27) (30) (32) (36)
DISCUSSION
The results of the experiments so far conducted appear to indicate that Nannochloris is a suitable food item for mass cultures. Its advantages over other organisms tested lie in its excellent adaptability to environmental conditions, as evidenced by its dominance in outdoor cultures throughout the year. The temporary contamination by Phaeoductylum and, to a lesser extent, Skeletonemu is caused by temperature changes. The same phenomenon is described by Goldman and Ryther (1976), Goldman (1977) and De Pauw et al. (1978b), for other phytoplankton mass cultures. An additional factor, which may favour the predominance of Nannochloris in our case, is the comparatively low salinity (average 15 o/oo) of the Baltic Sea water used for the cultures. We did not pay special attention to the concentration of silicates as a species determining factor. The agricultural fertilizer used contains 0.71% SiOZ by weight and the content of the sewage water is of the order of 30 mg/l. The sea water pumped to the culture site is filtered through a gravel bed, where it probably takes up silicates. We therefore do not think that silicon deficiency by preventing diatoms from becoming dominant, could be an important factor in our cultures.
179
The production and yield per m* during the year is comparable to other values reported for our latitude (Soeder, 1976; De Pauw et al., 1978b). We are aware of the harmful effects of the high pH values in our cultures which cause a growth inhibition due to lack of CO2 and bicarbonate. Other nutrients are lost by transformation of NH4+ into NH3 gas and precipitation of phosphate. Production could possibly be enhanced by blowing CO2 into the cultures, but this is merely an economic problem. Of particular relevance are the positive results obtained from feeding experiments with Brachionus and Eurytemora, both of which play an important role in our turbot larvae rearing project (Kuhlmann et al., 1981). Since we worked without pre-treated water, thus avoiding the high costs involved, our type of mass culture has the disadvantage of being susceptible to contamination by grazers, especially Brachionus, the ciliate Euplotes and various heterotrophic flagellates. A mass development of these grazers can destroy a culture within a few days. To avoid this, it is advisable to harvest the algae crop in time. In the case of small culture volumes, treatment with approximately 20 ppm of 40% formalin is fairly effective against ciliates and heterotrophic flagellates (Rothbard, 1975). For practical reasons we decided to work with semi-continuous cultures, harvesting not continuously but at intervals. Continuous cultures (chemostat or turbidostat) are superior in theory to these as far as production capacity is concerned, and can also be maintained in outdoor cultures of greater volume (Shelef et al., 1969; Ryther et al., 1975; De Pauw et al., 197813). In connection with feeding exact doses of different volumes from one algal tank to secondary producers in several tanks, it seems impractical to run a continuous flow culture. Despite this, it might be possible to run a combination of, say, an oyster culture together with an algal tank, suitable in volume to the consumers, as continuous cultures. The most economic fertilizer would be purified waste water (Goldman, 1976; Oswald et al., 1977). However, the quality fluctuates depending on the efficiency of the waste water plant. Moreover, there is again the danger of contamination by grazers. Toxic substances may be present and harm sensitive organisms. The more thoroughly the waste water is treated, the greater would be the quantity required on account of the lower nutrient content, the result being an undesirable dilution of the seawater. ACKNOWLEDGEMENTS
We are grateful to Dr. Koch, Pflanzenphysiologisches Institut der Universit& Gattingen for identifying the species and to Dipl.-Biol. Neudecker, Bundesforschungsanstalt fiir Fischerei, Hamburg, for providing the oyster spat. The study was financed by the Federal Ministry for Research and Technology (Project ‘Marine Bioproduction’ ME 0320/7).
180 REFERENCES Bartels, D., 1978. Untersuchung verschiedener Druckluftsysteme zur Turbulenzerzeugung in Algenzuchtbecken. Studienabschlussarbeit in der Fachrichtung Bioingenieurwesen, Fachhochschule Hamburg, 67 pp. Bienfang, P.K., 1975. Steady state analysis of nitrate-ammonium assimilation by phytoplankton. Limnol. Oceanogr., 20: 402-411. Briggs, R.P., 1978. Aspects of oyster culture in Strangford Lough, Northern Ireland. Aquaculture, 15: 307-318. Cummins, K.W. and Wuyceck, J.C., 1971. Caloric equivalents for investigations in ecological energetics. Mitt. Int. Ver. Limnol., 18: l-158. De Pauw, N., Bruggeman, E. and Persoone, G., 1978a. Research on tertiary treatment of swine manure by mass culturing of algae. Mitt. Int. Ver. Limnol., 21: 490-506. De Pauw, N., Verlet, H. and De Leenheer Jr., L., 1978b. Heated and unheated outdoor cultures of marine algae with animal manure. Presented at the First International Symposium on Production and Use of Microalgae Biomass, Acco, Israel, Sept. 1978. Epifanio, C.E. and Mootz, C.A., 1976. Growth of oysters in a recirculating maricultural system. Proc. Nat. Shellfish. Assoc., 66: 32-37. Eppley, R.W., Renger, E.H., Harrison, W.G. and Cullen, J.J., 1979. Ammonium distribution in southern California coastal waters and its role in the growth of phytoplankton. Limnol. Oceanogr., 24: 495-509. Furukawa, J. and Hidaka, K., 1973. Technical problems encountered in the mass culture of rotifer using marine yeast as food organisms. Bull. Plankton Sot. Japan, 20: 61-69. Goldman, J.C., 1976. Phytoplankton response to waste-water nutrient enrichment in continuous culture. J. Exp. Mar. Biol. Ecol., 23: 31-43. Goldman, J.C., 1977. Biomass production in mass cultures of marine phytoplankton at varying temperatures. J. Exp. Mar. Biol. Ecol., 27: 161-169. Goldman, J.C. and Ryther, J.H., 1976. Temperature-influenced species-competition in mass cultures of marine phytoplankton. Biotechnol. Bioeng., 18: 1125-1144. Grasshoff, K., 1976. Methods of Seawater Analysis. Chemie-Verlag, Weinheim-New York, 317 pp. Hirayama, K. and Ogawa, S., 1972. Fundamental studies on physiology of rotifer for its mass culture. I. Filter feeding of rotifer. Bull. Jap. Sot. Sci. Fish., 38: 1207-1214. Kuhlmann, D., Quantz, G. and Witt, U., 1981. Rearing of turbot larvae (Scophthalmus muximus L.) on cultured food organisms and post-metamorphosis growth on natural and artificial food. Aquaculture, 23: 183-196. Lenz, J., 1971. Zur Methode der Sestonbestimmung. Kieler Meeresforsch., 27: 180-193. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the folin-phenol reagent. J. Biol. Chem., 193: 265-275. McCarthy, J.J., Taylor, W.R. and Taft, J.L., 1977. Nitrogenous nutrition of the plankton in the Chesapeake Bay. 1. Nutrient availability and phytoplankton preference. Limnol. Oceangr., 22: 996-1011. Malouf, R.E. and Breese, W.P., 1977. Seasonal changes in the effects of temperature and water flow rate on the growth of juvenile Pacific oysters, Crassostrea gigas, Aquaculture, 12: l-13. Meixner, R., 1979. Culture of Pacific oysters (Crassostrea gigas) in containers in German coastal waters. In: T.V.R. Pillay and A. Dill (Editors), Advances in Aquaculture. Fishing News Book Ltd., Famham, Surrey, England, pp. 338-339. Oswald, W.J., Benemann, J.R. and Koopman, B.L., 1977. Production of biomass from fresh water aquatic systems using microalgae. Proc. Fuels from Biomass Symp., Univ. Illinois, Champaign, IL, pp. 59-81. Persoone, G. and Claus, C., 1978. Mass culturing of marine algae - a bottle-neck in the nursery culturing of molluscs. Presented at the First International Symposium on Production and Use of Microalgae Biomass, Acco, Israel, Sept. 1978.
181 Queisser, C., 1978. Untersuchungen iiber die Wachstumsrate und biochemische Zusammen setzung mariner Mikroalgen bei Abwasserzugabe als Diingemittel. Staatsexamensarbeit, Univ. Kiel, 88 pp. Rothbard, S., 1975. Control of Euplotes spec. by formalin in growth tanks of Chlorello spec. used as growth medium for the rotifer Brachonius plicatilis which serves as feed for hatchlings, Bamidgeh, 27: 100-109. Ryther, J.H., Goldman, J.C., Gifford, C.E., Huguenin, J.E., Wing, A.S., Clarner, J.P. Williams, L.D. and Lapointe, B.E., 1975. Physical models of integrated waste recycling marine polyculture systems. Aquaculture, 5: 163-177. Salser, B.R. and Mock, C.R., 1973. An air-lift circulator for algal culture tanks. In: J.W. Avault Jr. (Editor), Proceedings of the 4th Annual Workshop of the World Mariculture Society, Louisiana State Univ., Division of Continuing Education, Baton Rouge, LA, pp. 295-297. SchrSder, S., 1978. Untersuchung des Algenwachstum bei Verwendung verschiedener Druckluftsysteme zur Turbulenzerzeugung. Studienabschlussarbeit in der Fachrichtung Bioingenieurwesen, Fachhochschule Hamburg, 77 pp. Shelef, G., Oswald, W.J. and Golueke, C.G., 1969. The continuous culture of algal biomass on wastes. In: I. Malek, K. Beram, Z. Fencl, V. Munk, J. &i&a and H. Smrfkovl (Editors), Continuous Cultivation of Microorganisms. Academic Press, New York and London, pp. 601-629. Soeder, C.J., 1976. The technical production of microalgae and its prospects in marine aquaculture. In: K. Tiews (Editor), Fisheries Resources and their Management in Southeast Asia. German Foundation for International Development (DSE), Berlin and Federal Res. Bd. Fisheries, FAO, Rome, pp. 321-333. Sorgeloos, P., Persoone, G., De Winter, F., Bossuyt, E. and De Pauw, N., 1977. Air-water pumps as cheap and convenient tools for high density culturing of microscopic algae. Proceedings of the 8th Annual Workshop of the World Mariculture Society, San Jose (Costa Rica), January 9-13, 1977, pp. 173-183. UNESCO, 1966. Determination of Photosynthetic Pigments in Sea Water. Monographs on Oceanographic Methodology I. UNESCO, Paris, 66 pp. Walne, P.R., 1972. The influence of current speed, body size and water temperature on the filtration rate of five species of bivalves. J. Mar. Biol. Assoc. U.K., 52: 345-374.