Cultivation of the microalgae Chaetoceros gracilis to feed the rotifer Brachionus plicatilis

The microalgae Chaetoceros gracilis was cultivated to feed the rotifer Brachionus plicatilis. Microalgae and rotifers were grown separately, each for 10 days in a growing system volume of 62,5ml to 20L (1st phase). This was followed by 10 days in 250L (2nd phase), after 10 days in 1 500L (3rd phase). The growth rate exponential population at intervals of 24h for C. gracilis was 0,72, 0,30, and 0,28cells/ml to three phases of cultivation. Within 24h the arithmetic growth rate for the population of B. plicatilis was 330, 1 858 and 13 912rotifers/day in three phases of cultivation. Twenty-four hour intervals counts from the increasing number of organisms in the cultivation of microalgae, allows the prediction of a rotifer production necessary to feed a certain number organisms.

In the aquatic natural ecosystems the continuity of the species depends on the balance established between the different levels of the food plot.Thus, the development and survival of larvae and juvenile depends on the presence of organisms that make up the phytoplankton and zooplankton, who in turn occur in the presence of adequate nutrients (Nieves et al., 1996).
Commercial mariculture of many marine and fresh water organisms has been limited by several factors including inconsistent larvae production.This is partially due to the difficulty of producing great quantities of high-quality live foods, especially microalgae and rotifers (Horstmann, 1985;Fulks & Main, 1991).Valenzuela, Gendrop, Pérez and Wilburn (1999) mentions that the use of environment nutrients derived from fertilizers, nitrogen and phosphorous F/2 is a viable alternative to the cultivation of C. muelleri as food for larvae of Litopenaeus vannamei.The diatom, Chaetoceros gracilis (F.Schütt, 1895), is extensively used as a nutritive food organism in the rearing of prawn larvae (Chu, 1989).Two of the best microalgae for aquaculture feeds are Isochrysis galbana and C. gracilis.Helm, Bourne and Lovatelli (2004) mentioned that among some cultured phytoplankton species used in bivalve mollusk and fish hatcheries is C. gracilis.Their effectiveness is due in part to their small size and n-3 HUFA content (Barclay & Zeller, 1996).
Rotifers fed on microalgae such as C. gracilis showed better viability, larger size and low ciliate contamination (Knu, 2004).
The purpose of this study was to culture up to 1 500L of the microalgae C. gracilis to feed 1 500L culture of rotifers (Brachionus plicatilis) (Müller, 1786).For subsequent use as live food for different organisms.

MATERIALS AND METHODS
Culture of C. gracilis and B. plicatilis (Tables 1 and 2) was initially indoors in sea water at 24°C (±1) and aerated by an electric pump.Volume was increased over the course of 10 days at 24h intervals increase, from 60,5ml to 20L under continuous artificial light from twelve 30 w fluorescent tubes.Two subsequent stages, held at 250L and 1 500L respectively, were outdoors, with 13h natural light and 11h darkness, which consisted of EDTA-Fe together with a metal solution Co, Zn, Cu, Mo (Nutrafin Plant Gro).
For C. gracilis, these subsequent stages used medium F as described by Guillard (1973), and followed the batch culture method (Coutteau, 1996).Natural sea water was filtered through 5μm then 1μm; then 0,25ml of commercial chlorine for 24h was used, and 0,25ml of sodium thiosulfate at 10% normal solution was applied to neutralize the commercial chlorine.
A number of the culture volume of microalgae harvested from 20L was used to seed the 250L volume, after 10 days a number of the microalgae harvested were used to seed the 1 500L volumes, and after 10 days these microalgae harvested were used to feed the rotifers (Table 1).At 24h intervals, a microalgae sample (1-10ml) was preserved with lugol and counted under a light microscope with a Neubauer hematocimeter to calculate the concentration (Odum, 1972).Population growth rate was calculated by the least-square method using the following formula: Number of cells = a e b (d) , where a is a constant, b is the slope, and d is duration of culture (days).
Culture of B. plicatilis was similar to that of the microalgae (Table 2).Rotifers were cultured in sterile sea water and fed at 24h intervals with at 1 x 106cells/ml microalgae.The total number of rotifers (nr) was calculated by adding the total number of rotifers to the total number of eggs/ml (nr = nr + ne) (Ramírez-Sevilla, 1991).Population growth (ml) was determined at 24h interval counts of 10-20 groups of rotifers.Daily population growth rate was calculated by the least-squared method using the following formula: Number of rotifers = a + b (d) where (d) is duration of culture (days), a is constant, and b is slope.

RESULTS
Table 1 shows that in 62,5ml the total average cells/ml initiated with 1 800 000 and finalized in 20L with 3 283 300cells/ml.In 250L initiated with 250 000 and it finalized with 3 866 700cells/ml.In 1 500L initiated with 200 000 and finalized with 2 850 000cells/ml.Table 2 shows that in 62,5ml the total average rotifers initiated with 35,7 and finalized in 20L with 151rotifers/ml.In 250L initiated with 42,7 and it finalise with 120rotifers/ml.In 1 500L initiated with 34,7 and finalized with 119rotifers/ml.
In general, population growth rate was exponential for microalgae and arithmetic for rotifers (Table 3).Also at growing volume culture of 62,5ml to 20L at 24h intervals exponential population growth rate could be expressed as 0,84 since the determination coefficient was 0,9.

DISCUSSION
When Vega-Pérez (1991), Sapién and Leal (1992) used Guillard's F medium, in a continuous culture of Tetraselmis suesica and Chaetoceros sp., they found that the production of microalgae was proportionally quantitative to the quantity of supplied nutrients.In present paper the microalgae growth rate results were similar.Also Reyes-Bustamante (1999), found that F medium obtained the highest yields of the microalgae Kirchneriella obesa, Scenedesmus quadricauda, and Chlorococcum infusorium, although its cost was a limiting factor.The rate of microalgae population growth, in media based on commercial agricultural fertilizers is comparable to the results with the F medium, but at lower cost.Use of F medium for microalgae culture enhances exponential without any toxic effects, since the constituent metals are necessary for synthesis of enzymatic growth cofactors used in oxy-reduction cycles (Round, 1973;Fogg, 1975).
Density of rotifers obtained in the present study was lower than those observed by other researchers (Hirata, 1980;Schluter & Groeneweg, 1981;Meralgemene, 1985;Hirayama, 1987).Maeda and Hino (1991) also mentioned  Once the production of microalgae is continuous, production of rotifers is continuous, and it is possible to feed larvae, reduce their natural mortality, and predict the number of larvae that will grow.

TABLE 1
Growth of the population C. gracilis cultivated for three consecutive periods of 10 days

TABLE 2
Population growth of B. plicatilis cultivated during three consecutive periods of 10 days that bacteria and protozoa are important biotic factors that affect the rate of growth of the rotifer population.

TABLE 3
Regression parameters of population growth of C. gracilis and B. plicatilis, at three culture volumes