Kenji Yoshimura1, Koji Usuki2, Takao Yoshimatsu2, Chikara Kitajima2 & Atsushi Hagiwara3. 1Fukuoka Mariculture Corporation, Fukuoka 811-35, Japan.
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Hydrobiologia 358: 139–144, 1997.
A. Hagiwara, T. W. Snell, E. Lubzens & C. S. Tamaru, Live Food in Aquaculture. c 1997 Kluwer Academic Publishers. Printed in Belgium.
Recent development of a high density mass culture system for the rotifer Brachionus rotundiformis Tschugunoff Kenji Yoshimura1 , Koji Usuki2, Takao Yoshimatsu2, Chikara Kitajima2 & Atsushi Hagiwara3 1
Fukuoka Mariculture Corporation, Fukuoka 811-35, Japan Fishery Research Laboratory, Kyushu University, Tsuyazaki, Fukuoka 811-33, Japan 3 Faculty of Fisheries, Nagasaki University, Bunkyo, Nagasaki 852, Japan 2
Key words: rotifer, Brachionus rotundiformis, live food, high density mass culture, packed volume, filtering equipment
Abstract Feeding rotifer mass cultures condensed Chlorella made it possible to culture rotifers at 103 ind. ml,1 Subsequently, it was realized that to raise the culture density of rotifers to 104 ind. ml,1 , inhibitory factors (low dissolved oxygen, foaming separation, and NH3 -N toxicity) needed to be reduced through oxygen gas supplementation and regulation of pH at 7. However, even after these improvements, problems remained to be solved. One is controlling debris, particulate organic matter and microbes which often clog the collection nets during harvest. Another problem is the development of a more accurate quantitative method for determining Chlorella and rotifer densities that could replace the conventional counting method. These problems have been resolved in the following manner. (1) Filtering equipment for removing particulate debris in the culture media: Filtering equipment made of a nylon mat and a stainless steel frame was developed to increase the surface area for debris removal. Using this filter, the harvest of high density culture at 104 rotifers ml,1 was possible without clogging of the collection net. (2) Quantitative determination of rotifers by a centrifugation method: We determined the abundance of rotifers by centrifuging samples and measuring their packed volume (PV, ml l,1 ). PV of rotifers was easier to and more accurate to measure (coefficient of variation, 4%) than a direct count of the density (coefficient of variation, 15%). Organic wastes in rotifer cultures made the measurement of rotifer PV difficult. By placing a filter in the mass culture tank, however, the boundary between rotifers and other organic wastes in a centrifuge tube was easily visualized. Introduction Mass culture of the marine rotifer Brachionus rotundiformis at ultra-high densities (exceeding 10 000 ind. ml,1 ) became possible in 1 m3 tank, firstly due to the introduction of a condensed phytoplankton diet that enabled to provide ample amount of food to rotifer biomass. Subsequent problems such as the shortage of oxygen and increase of free ammonia was solved by supplying oxygen gas and by regulating pH. Such progress including schematic design and operation cost of the system was reviewed by Yoshimura et al. (1996). In order to make this culture system practically feasible, a filter was required to remove the large amount
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of organic waste accumulating in the rotifer cultures, since this disturbs the rotifer harvest and feeding of fish larvae. Organic wastes may also promote the growth of pathogens that could cause fish diseases. Such wastes are mainly composed of rotifer feces, amictic egg shells, and probionts including bacteria, protozoa and fungi. To overcome this problem, we invented a filtration device to eliminate suspended matter other than the rotifers from the culture water. Another goal was to establish a method to quantify rotifer biomass in the high density cultures. Typical counting methods have a large variation and low precision. Coefficients of variation as high as 20% have been reported (Hirano, 1986). For high density cultures
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140 at 104 rotifers ml,1 in 1 m3 tanks, counting methods have the possibility to result in errors of on the order of 109 . It also requires sample dilution and counting procedures. To solve these problems, we tested the effectiveness of using the packed volume method to measure the rotifer biomass. This method is commonly used to estimate the amount of blood cells or phytoplankton. We also obtained estimates of the amount of organic wastes in the culture by packed volume, so that the effect of the filtration system could be quantified. This paper summarizes our achievements made during the last two years.
Materials and methods In all experiments, we used the Fukuoka strain of Brachionus rotundiformis. This strain has been cultured for larval rearing practices at Fukuoka Mariculture Corporation since 1991. The mean standard deviation of lorica length is 190 14 m (n = 50). Condensed Chlorella regularis (Nisshin Science Co. Ltd.; average cell density, 150 108 cells ml,1 ; PCV, 600 ml l,1 ) was continuously fed to rotifers using a peristaltic pump delivering 0.5 l condensed Chlorella suspension per 108 rotifers per day. During the rotifer culture period, oxygen gas was supplied at 1–5 l min,1 , and pH was adjusted to 7 by adding hydrochloric acid. Salinity and temperature were regulated at 33 ppt and 32 C, respectively. Other details of culture methods can be found in Yoshimura et al., 1996.
Results Quantitative determination of rotifers by the centrifuging method Table 1 compares estimates of rotifer and Chlorella densities obtained by counting and centrifugation methods. By sieving and concentrating clean rotifer cultures, a rotifer sample without the visible contamination of organic particulate matters was prepared and used as a control. A Chlorella sample was prepared by diluting the paste one hundred times. The samples were transferred to a glass 0.03 ml hematocrit tube (Nakazono Instrument Co., Ltd.) using a 1.0 ml digital pipette (Gilson, accuracy of 1.0%). The hematocrit tube has 0.001 ml gradations. Chlorella was centrifuged at 2800 g for 30 min (Kokusan H-103N, rotor diameter – 31.4 cm) following the rec-
ommendation by Aruga (1979). From a series of experiments, we also confirmed that the packed volume of rotifers was constant after centrifugation at 2800 g for 30 min. When rotifer density exceeded 104 ind. ml,1 , approximately 1.0 ml sample was necessary to obtain a stable packed volume with less than a 5% coefficient of variation (c.v.). Data of Chlorella and rotifer biomass from both methods had a normal distribution. The c.v. of the counting method, however, was 15.3%, which was 4.2 times larger than the centrifugation method (c.v. = 3.6%). From these data, it was estimated that the counting method requires 31 and 36 l ml samples for Chlorella and rotifers, respectively, to obtain a 95% confidence interval that will not be wider than 5% of the population mean. With the centrifugation method, however, only 3 samples are sufficient both for Chlorella and rotifers. There was a problem, however, when samples from the high density mass cultures of rotifers were directly centrifuged. Some of the upper layer components (mainly larger sized particulate organic matter) penetrated into the rotifer layer, making quantification of rotifer PV difficult because the boundary of two layers was not distinct. Filter equipment was originally invented to simplify rotifer harvest by removing excess organic wastes from high density rotifer cultures. But this filter also permitted accurate quantification of rotifer PV. When filters were placed in the rotifer culture, and samples were centrifuged, the pelleted material was clearly separated into three layers (Figure 1, left). From the bottom, a brown colored layer (composed of rotifers and amictic eggs), a green layer (composed of organic wastes, such as undigested Chlorella, empty egg shells, rotifer feces and protozoa), and a white layer (composed of bacteria and fungi identified with a compound microscopy) were visible. Thus, by placing the filter in the rotifer mass culture tank, it became possible to measure rotifer PV as well as the amount of organic waste. This was probably because the filter removed larger particles of organic wastes without removing rotifers. The PV volume of the brown layer was significantly correlated with rotifer density obtained by the counting method (Y = 0.0009X, r2 = 0.61, P
