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Culture of Vibrio alginolyticus C7b, a potential probiotic bacterium, with the microalga Chaetoceros muelleri
Aquaculture 211 (2002) 43 – 48 www.elsevier.com/locate/aqua-online
Culture of Vibrio alginolyticus C7b, a potential probiotic bacterium, with the microalga Chaetoceros muelleri B. Gomez-Gil *, A. Roque, G. Velasco-Blanco CIAD/Mazatla´n Unit for Aquaculture and Environmental Management, AP. 711 Mazatla´n, Sinaloa Mexico CP 82000, Mexico Received 12 March 2001; received in revised form 27 December 2001; accepted 27 December 2001
Abstract In this study, an attempt is made to evaluate the performance of the microalga Chaetoceros muelleri when cultured with a potential probiotic bacterium Vibrio alginolyticus strain C7b as compared when both are cultured alone in medium f/2.Strain C7b grew significantly better and lasted longer when grown with the microalga than when grown alone. The microalga density was not affected by the presence of the bacteria compared when grown alone.C. muelleri and the bacterial strain C7b can be cultured together for up to 9 days to achieve a high density (5.15106 and 6.63104 cell ml 1, respectively) and then fed to the protozoeal and mysis stages of penaeid shrimp. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Probiotics; Bacteria; Microalga; Chaetoceros muelleri; Vibrio alginolyticus; Shrimp larvae
1. Introduction Probiotic microorganisms have been used to enhance the resistance to diseases of aquatic animals with variable results (Griffith, 1995; Riquelme et al., 1997; Gibson et al., 1998; Gomez-Gil et al., 2000). Vibrio alginolyticus has been tested as a probiotic in Litopenaeus vannamei larvae with promising results, and it was found to give some protection against disease (Austin et al., 1995; Garriques and Arevalo, 1995). V. alginolyticus was also detected
* Corresponding author. Fax: +00-52-669-9880159. E-mail address: [email protected] (B. Gomez-Gil). 0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 0 0 4 - 2
44
B. Gomez-Gil et al. / Aquaculture 211 (2002) 43–48
in healthy rotifers and a positive relation between the survival of turbot and the presence of this vibrio in the rearing environment was established (Gatesoupe, 1990). A potential probiotic strain, coded C7b and identified as V. alginolyticus, proved to be able to outgrow several other bacterial strains isolated from seawater when a bioassay was done employing a technique based on the disk diffusion method (Gomez-Gil, 1998). This strain also demonstrated to be effective in overgrowing potential pathogenic vibrios isolated from the haemolymph of diseased shrimps and had no detrimental effect to the shrimp larvae. Species of the microalgae Chaetoceros have been used widely to feed larval stages of penaeid shrimp, specially the protozoeas. Commonly, the algal density fed to the shrimp larvae is around 105 cells ml 1 (Wyban and Sweeney, 1991), but varies with the larval stage. Microalgae employed in shrimp hatcheries usually have a natural bacterial load between 104 and 107 CFU ml 1 of heterotrophic bacteria but rarely of vibrios (Liza´rragaPartida et al., 1997). Improvements in the growth of some microalgae has been demonstrated when bacteria are present. Enhanced growth of the microalga Tetraselmis chuii was obtained when cultured with bacteria commonly found in its culture (Canizares-Villanueva and Ontiveros-Arredondo, 1993). The same effect was observed for C. gracilis when grown with Flavobacterium sp. where significant improvement in the specific growth rate of the microalga was obtained and the stationary growth phase lasted longer (Suminto and Hirayama, 1997). However, these authors did not find the same improvement for Isochrysis galbana or Pavlova lutheri, which point out to a species-specific relationship. Growing bacteria in volumes of several liters without contamination is difficult to accomplish in shrimp hatcheries where sterile conditions are hard to maintain. Therefore, if probiotic bacteria can be cultured with the microalga in the same container, the work of feeding the larvae can be much simplified. The main objective of this work is to evaluate the performance of the culture of a potential probiont (C7b) with a microalga commonly used in shrimp larval culture, as a mean to maintain a more stable and longer lasting culture of the bacterium.
2. Materials and methods The potential probiotic bacterium coded C7b was isolated from seawater in Mazatla´n, ´ Mexico and was previously characterized (Gomez-Gil, 1998). Relevant characteristics of this isolate were: it grew as a yellow colony on thiosulphate citrate bile-salt sucrose agar (TCBS, Difco, USA) and swarmed in Marine Agar (Bioxon, Me´xico). It was motile and fermented glucose, sensitive to 0/129 10 and 150 Ag, and positive for oxidase (Difco), gelatinase (Kohn), D-mannitol (Bioxon), D-mannose (Difco), sucrose (Difco), L-leucine (Sigma, Germany) but negative for cellobiose (Sigma), h-hydroxybutyrate (Sigma), lactose (Difco) and arginine dehydrolase (Sigma). These properties identify isolate C7b as V. alginolyticus (Baumann and Schubert, 1983). The microalga employed was Chaetoceros muelleri (CHM1) obtained from CICESE, Mexico. It was cultured in f/2 medium (Guillard and Ryther, 1962) at 20F1 jC with constant illumination with 39 W fluorescent lamps (0.121017 quanta s 1 cm 1). The microalga
B. Gomez-Gil et al. / Aquaculture 211 (2002) 43–48
45
stock cultures were maintained in 20 ml test tubes with f/2 medium and were agitated manually every day for up to 10 days. Each tube was used to inoculate a 600 ml erlenmeyer flask or a five-port glass fermentor with f/2 medium. In these volumes, aeration was provided through a 0.45 Am filter connected to a 2.5 HP air blower (Siemmens). 2.1. Bacterial culture and preparation of the inocula The bacterial isolate was preserved at preserved at 70 jC as recommended by Gherna (1994). It was recovered from the ultra-low freezer and inoculated into Tryptic Soy Broth (TSB, Difco) supplemented with 2.0% NaCl, then incubated at 30 jC for 20– 24 h. Ten milliliters of the broth were centrifuged (Hereaus model Biofuge Primo R) in Oak ridge sterile centrifuge tubes of 10 ml at 6139g for 10 min at 10 jC, after which the supernatant was discharged and the bacterial pellet suspended in sterile saline solution (2.5%). This process was repeated and the cell density was adjusted to an optical density of 1.0 at 610 nm in a spectrophotometer (Hach DR 2000, Hach, Loveland). This suspension was equal to 0.5 on the McFarland scale (approximately 1.0108 CFU ml 1). To estimate the bacterial density in the system, the suspension was serially diluted in sterile saline solution and 100 Al were spreaded onto Tryptic Soy Agar (TSA, Difco) supplemented with 2.0% NaCl. 2.2. Experimental design Three treatments were conducted: microalga alone, C7b alone, and microalga and C7b together. All the treatments were conducted in a five-port glass fermentor containing 600 ml of f/2 medium. One milliliter of the bacterial culture (approximately 6.0107 CFU ml 1) was inoculated in the fermentor to obtain a final density of 1.0105 CFU ml 1. The growth conditions were the same as those described previously for microalga culture. Each experiment was performed in triplicate and the density of microalga and bacteria evaluated daily. The microalga were counted in duplicate samples with a haemocytometer (Hausser Scientific, USA) and the bacteria were evaluated by viable total count on Marine and TCBS agars. The pH of the water was also recorded daily.
3. Results The results of the experiments show that isolate C7b grew to significantly higher densities and maintained the high density for longer when cultured with the microalga than when cultured alone in the f/2 medium (Fig. 1). When cultured together, the bacterial density was maintained almost without significant change during the 9 days of the incubation period, whereas when cultured alone, the density of C7b decreased after the first 24 h. At day 1, no significant difference was observed in the density of C7b in both treatments (t-test= 1.578, p=0.190, n=3). At day 9, significant differences were obtained (t-test=4.112, p=0.015, n=3); the values were 4.53103 cell ml 1 when cultured alone and 6.63104 cell ml 1 when cultured together. The microalga density was not affected by the presence of the bacteria (Fig. 2) compared when grown alone. At the end of the incubation period (9 days), no significant
46
B. Gomez-Gil et al. / Aquaculture 211 (2002) 43–48
Fig. 1. Mean bacterial density of isolate C7b when cultured alone and when cultured together with the microalga C. muelleri. Error bars represent the standard deviation of the mean.
difference was observed in the microalga density when cultured alone than when cultured together (t-test=0.987, p=0.380, n=3); the values were 3.57106 and 5.15106 cell ml 1, respectively. In all the experiments, there was a significant presence of marine bacteria (not identified) other than vibrios and their variation during the experimental period was very
Fig. 2. Mean density of the microalga C. muelleri when cultured alone and when cultured together with the bacterium C7b. Error bars represent the standard deviation of the mean.
B. Gomez-Gil et al. / Aquaculture 211 (2002) 43–48
47
high. Their density did not seem to be affected by the presence of the microalga, the strain C7b or both (data not shown). The mean pH for the microalga alone treatment for all the days was 8.45 (SD 0.306, max 8.89, min 7.97, n=8 days), for the C14 alone was 7.95 (SD 0.067, max 8.03, min 7.83, n=9 days), and for the treatment where both were cultured was 8.16 (SD 0.159, max 8.32, min 7.88, n=9 days). No significant differences were observed in the pH values between the days that the experiment lasted for the microalga treatment (ANOVA=0.102, p=0.327, n=3, 8 days) and for the C7b treatment (ANOVA=0.472, p=0.054, n=3, 9 days). When microalga and C7b were cultured together, significant differences were obtained between the days ( p<0.001). The pH descended to a minimum at day 3 and then began to rise to a maximum by days 8 and 9.
4. Discussion In this study, it was shown that a potential probiotic bacterium could be cultured in the same system as the microalga without detriment to the yield of either species. The Vibrio strain C7b performed better when cultured with C. muelleri than when alone. Other microalgae species have been observed to perform better when cultured with bacteria than when in an axenic culture (Canizares-Villanueva and Ontiveros-Arredondo, 1993; Suminto and Hirayama, 1997). A synergistic effect might be dependent on the species of microalga and bacteria tested, and no generalizations can be drawn. Enhancement in the growth of C7b could be attributed to the production of nutrients by C. muelleri or by the high organic material available when the microalgal cells dies. Strain C7b was found to be of no danger to the survival and performance of penaeid larvae, even at densities of up to 7.1104 CFU ml 1 in the rearing tanks (Gomez-Gil, 1998). The presence of other heterotrophic bacteria in the microalga culture did not affect the performance of strain C7b. Rico-Mora et al. (1998) found that this effect was not observed when Skeletonema costatum was cultured with V. alginolyticus and a bacterium isolated from normal microalga cultures (SK-05, probably an Aeromonas); SK-05 out-competed the vibrio, reducing its numbers 10-fold. The density of V. alginolyticus obtained by the previous authors was 6.8105 CFU ml 1 whereas in this study, the maximum density obtained was 9.6104 CFU ml 1. The higher number of V. alginolyticus found by other authors might be attributed to the species of microalga employed since the culture conditions were similar. With data presented here, it can be suggested that C. muelleri and the bacterial strain C7b can be cultured together for up to 9 days to achieve a density of 5.15106 and 6.63104 cell ml 1, respectively, and then fed to the protozoeal and mysis stages of penaeid shrimp.
Acknowledgements Carmen Bola´n Mejı´a. This work was financed by the International Foundation for Science project A/2203-2.
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B. Gomez-Gil et al. / Aquaculture 211 (2002) 43–48
References Austin, B., Stuckey, F., Robertson, P., Effendi, I., Griffiths, D., 1995. A probiotic strain of Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalli. J. Fish Dis. 18, 93 – 96. Baumann, P., Schubert, R.H.W., 1983. Vibrionaceae. In: Krieg, N.R., Holt, G.J. (Eds.), Bergey’s Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, pp. 516 – 550. Canizares-Villanueva, A., Ontiveros-Arredondo, C., 1993. Comportamiento cine´tico de un cultivo mixto de la microalga marina Tetraselmis chuii y dos bacterias. Rev. Invest. Mar. 14, 86 – 91. Garriques, D., Arevalo, G., 1995. An evaluation of the production and use of a live bacterial isolate to manipulate the microbial flora in the commercial production of Penaeus vannamei postlarvae in Ecuador. In: Browdy, C.L., Hopkins, J.S. (Eds.), Swimming Through Troubled Water. Proceedings of the Special Session on Shrimp Farming, Aquaculture ’95. World Aquaculture Society, Baton Rouge, pp. 53 – 59. Gatesoupe, F., 1990. The continuous feeding of turbot larvae, Scophthalmus maximus, and control of the bacterial environment of rotifers. Aquaculture 89, 139 – 148. Gherna, L.R., 1994. Culture preservation. In: Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R. (Eds.), Methods for General and Molecular Microbiology. American Society for Microbiology, Washington, pp. 278 – 292. Gibson, L.F., Woodworth, J., George, A.M., 1998. Probiotic activity of Aeromonas media on the Pacific oyster, Crassostrea gigas, when challenged with Vibrio tubiashii. Aquaculture 169, 11 – 120. Gomez-Gil, B., 1998. Evaluation of potential probionts for use in a penaeid shrimp larval culture. PhD Thesis, The University of Stirling, 269 pp. Gomez-Gil, B., Roque, A., Turnbull, J., 2000. The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. Aquaculture 191, 259 – 270. Griffith, D.R.W., 1995. Microbiology and the role of probiotics in Ecuadorian shrimp hatcheries. In: Lavens, P., Jaspers, E., Roelands, I. (Eds.), Larvi ’91—Fish and Crustacean Larviculture Symposium. European Aquaculture Society, Gent, p. 478, Special publication no. 24. Guillard, R., Ryther, J., 1962. Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8, 229 – 239. Liza´rraga-Partida, M., Montoya-Rodrı´guez, L., Gentrop-Funes, V., 1997. The use of bacterial counts in two Mexican shrimp hatcheries. Cienc. Mar. 23, 129 – 140. Rico-Mora, R., Voltolina, D., Villaescusa-Celaya, J., 1998. Biological control of Vibrio alginolyticus in Skeletonema costatum (Bacillariophyceae) cultures. Aquacult. Eng. 19, 1 – 6. Riquelme, C., Araya, R., Vergara, N., Rojas, A., Guaita, M., Candia, M., 1997. Potential probiotic strains in the culture of the Chilean scallop Argopecten purpuratus (Lamarck, 1819). Aquaculture 154, 17 – 26. Suminto, A., Hirayama, K., 1997. Application of a growth-promoting bacteria for stable mass culture of three marine microalgae. Hydrobiologia 358, 223 – 230. Wyban, J.A., Sweeney, J.N., 1991. Intensive Shrimp Production Technology. The Oceanic Institute, Honolulu, 185 pp.
Culture of Vibrio alginolyticus C7b, a potential probiotic bacterium, with the microalga Chaetoceros muelleri B. Gomez-Gil *, A. Roque, G. Velasco-Blanco CIAD/Mazatla´n Unit for Aquaculture and Environmental Management, AP. 711 Mazatla´n, Sinaloa Mexico CP 82000, Mexico Received 12 March 2001; received in revised form 27 December 2001; accepted 27 December 2001
Abstract In this study, an attempt is made to evaluate the performance of the microalga Chaetoceros muelleri when cultured with a potential probiotic bacterium Vibrio alginolyticus strain C7b as compared when both are cultured alone in medium f/2.Strain C7b grew significantly better and lasted longer when grown with the microalga than when grown alone. The microalga density was not affected by the presence of the bacteria compared when grown alone.C. muelleri and the bacterial strain C7b can be cultured together for up to 9 days to achieve a high density (5.15106 and 6.63104 cell ml 1, respectively) and then fed to the protozoeal and mysis stages of penaeid shrimp. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Probiotics; Bacteria; Microalga; Chaetoceros muelleri; Vibrio alginolyticus; Shrimp larvae
1. Introduction Probiotic microorganisms have been used to enhance the resistance to diseases of aquatic animals with variable results (Griffith, 1995; Riquelme et al., 1997; Gibson et al., 1998; Gomez-Gil et al., 2000). Vibrio alginolyticus has been tested as a probiotic in Litopenaeus vannamei larvae with promising results, and it was found to give some protection against disease (Austin et al., 1995; Garriques and Arevalo, 1995). V. alginolyticus was also detected
* Corresponding author. Fax: +00-52-669-9880159. E-mail address: [email protected] (B. Gomez-Gil). 0044-8486/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 0 0 4 - 2
44
B. Gomez-Gil et al. / Aquaculture 211 (2002) 43–48
in healthy rotifers and a positive relation between the survival of turbot and the presence of this vibrio in the rearing environment was established (Gatesoupe, 1990). A potential probiotic strain, coded C7b and identified as V. alginolyticus, proved to be able to outgrow several other bacterial strains isolated from seawater when a bioassay was done employing a technique based on the disk diffusion method (Gomez-Gil, 1998). This strain also demonstrated to be effective in overgrowing potential pathogenic vibrios isolated from the haemolymph of diseased shrimps and had no detrimental effect to the shrimp larvae. Species of the microalgae Chaetoceros have been used widely to feed larval stages of penaeid shrimp, specially the protozoeas. Commonly, the algal density fed to the shrimp larvae is around 105 cells ml 1 (Wyban and Sweeney, 1991), but varies with the larval stage. Microalgae employed in shrimp hatcheries usually have a natural bacterial load between 104 and 107 CFU ml 1 of heterotrophic bacteria but rarely of vibrios (Liza´rragaPartida et al., 1997). Improvements in the growth of some microalgae has been demonstrated when bacteria are present. Enhanced growth of the microalga Tetraselmis chuii was obtained when cultured with bacteria commonly found in its culture (Canizares-Villanueva and Ontiveros-Arredondo, 1993). The same effect was observed for C. gracilis when grown with Flavobacterium sp. where significant improvement in the specific growth rate of the microalga was obtained and the stationary growth phase lasted longer (Suminto and Hirayama, 1997). However, these authors did not find the same improvement for Isochrysis galbana or Pavlova lutheri, which point out to a species-specific relationship. Growing bacteria in volumes of several liters without contamination is difficult to accomplish in shrimp hatcheries where sterile conditions are hard to maintain. Therefore, if probiotic bacteria can be cultured with the microalga in the same container, the work of feeding the larvae can be much simplified. The main objective of this work is to evaluate the performance of the culture of a potential probiont (C7b) with a microalga commonly used in shrimp larval culture, as a mean to maintain a more stable and longer lasting culture of the bacterium.
2. Materials and methods The potential probiotic bacterium coded C7b was isolated from seawater in Mazatla´n, ´ Mexico and was previously characterized (Gomez-Gil, 1998). Relevant characteristics of this isolate were: it grew as a yellow colony on thiosulphate citrate bile-salt sucrose agar (TCBS, Difco, USA) and swarmed in Marine Agar (Bioxon, Me´xico). It was motile and fermented glucose, sensitive to 0/129 10 and 150 Ag, and positive for oxidase (Difco), gelatinase (Kohn), D-mannitol (Bioxon), D-mannose (Difco), sucrose (Difco), L-leucine (Sigma, Germany) but negative for cellobiose (Sigma), h-hydroxybutyrate (Sigma), lactose (Difco) and arginine dehydrolase (Sigma). These properties identify isolate C7b as V. alginolyticus (Baumann and Schubert, 1983). The microalga employed was Chaetoceros muelleri (CHM1) obtained from CICESE, Mexico. It was cultured in f/2 medium (Guillard and Ryther, 1962) at 20F1 jC with constant illumination with 39 W fluorescent lamps (0.121017 quanta s 1 cm 1). The microalga
B. Gomez-Gil et al. / Aquaculture 211 (2002) 43–48
45
stock cultures were maintained in 20 ml test tubes with f/2 medium and were agitated manually every day for up to 10 days. Each tube was used to inoculate a 600 ml erlenmeyer flask or a five-port glass fermentor with f/2 medium. In these volumes, aeration was provided through a 0.45 Am filter connected to a 2.5 HP air blower (Siemmens). 2.1. Bacterial culture and preparation of the inocula The bacterial isolate was preserved at preserved at 70 jC as recommended by Gherna (1994). It was recovered from the ultra-low freezer and inoculated into Tryptic Soy Broth (TSB, Difco) supplemented with 2.0% NaCl, then incubated at 30 jC for 20– 24 h. Ten milliliters of the broth were centrifuged (Hereaus model Biofuge Primo R) in Oak ridge sterile centrifuge tubes of 10 ml at 6139g for 10 min at 10 jC, after which the supernatant was discharged and the bacterial pellet suspended in sterile saline solution (2.5%). This process was repeated and the cell density was adjusted to an optical density of 1.0 at 610 nm in a spectrophotometer (Hach DR 2000, Hach, Loveland). This suspension was equal to 0.5 on the McFarland scale (approximately 1.0108 CFU ml 1). To estimate the bacterial density in the system, the suspension was serially diluted in sterile saline solution and 100 Al were spreaded onto Tryptic Soy Agar (TSA, Difco) supplemented with 2.0% NaCl. 2.2. Experimental design Three treatments were conducted: microalga alone, C7b alone, and microalga and C7b together. All the treatments were conducted in a five-port glass fermentor containing 600 ml of f/2 medium. One milliliter of the bacterial culture (approximately 6.0107 CFU ml 1) was inoculated in the fermentor to obtain a final density of 1.0105 CFU ml 1. The growth conditions were the same as those described previously for microalga culture. Each experiment was performed in triplicate and the density of microalga and bacteria evaluated daily. The microalga were counted in duplicate samples with a haemocytometer (Hausser Scientific, USA) and the bacteria were evaluated by viable total count on Marine and TCBS agars. The pH of the water was also recorded daily.
3. Results The results of the experiments show that isolate C7b grew to significantly higher densities and maintained the high density for longer when cultured with the microalga than when cultured alone in the f/2 medium (Fig. 1). When cultured together, the bacterial density was maintained almost without significant change during the 9 days of the incubation period, whereas when cultured alone, the density of C7b decreased after the first 24 h. At day 1, no significant difference was observed in the density of C7b in both treatments (t-test= 1.578, p=0.190, n=3). At day 9, significant differences were obtained (t-test=4.112, p=0.015, n=3); the values were 4.53103 cell ml 1 when cultured alone and 6.63104 cell ml 1 when cultured together. The microalga density was not affected by the presence of the bacteria (Fig. 2) compared when grown alone. At the end of the incubation period (9 days), no significant
46
B. Gomez-Gil et al. / Aquaculture 211 (2002) 43–48
Fig. 1. Mean bacterial density of isolate C7b when cultured alone and when cultured together with the microalga C. muelleri. Error bars represent the standard deviation of the mean.
difference was observed in the microalga density when cultured alone than when cultured together (t-test=0.987, p=0.380, n=3); the values were 3.57106 and 5.15106 cell ml 1, respectively. In all the experiments, there was a significant presence of marine bacteria (not identified) other than vibrios and their variation during the experimental period was very
Fig. 2. Mean density of the microalga C. muelleri when cultured alone and when cultured together with the bacterium C7b. Error bars represent the standard deviation of the mean.
B. Gomez-Gil et al. / Aquaculture 211 (2002) 43–48
47
high. Their density did not seem to be affected by the presence of the microalga, the strain C7b or both (data not shown). The mean pH for the microalga alone treatment for all the days was 8.45 (SD 0.306, max 8.89, min 7.97, n=8 days), for the C14 alone was 7.95 (SD 0.067, max 8.03, min 7.83, n=9 days), and for the treatment where both were cultured was 8.16 (SD 0.159, max 8.32, min 7.88, n=9 days). No significant differences were observed in the pH values between the days that the experiment lasted for the microalga treatment (ANOVA=0.102, p=0.327, n=3, 8 days) and for the C7b treatment (ANOVA=0.472, p=0.054, n=3, 9 days). When microalga and C7b were cultured together, significant differences were obtained between the days ( p<0.001). The pH descended to a minimum at day 3 and then began to rise to a maximum by days 8 and 9.
4. Discussion In this study, it was shown that a potential probiotic bacterium could be cultured in the same system as the microalga without detriment to the yield of either species. The Vibrio strain C7b performed better when cultured with C. muelleri than when alone. Other microalgae species have been observed to perform better when cultured with bacteria than when in an axenic culture (Canizares-Villanueva and Ontiveros-Arredondo, 1993; Suminto and Hirayama, 1997). A synergistic effect might be dependent on the species of microalga and bacteria tested, and no generalizations can be drawn. Enhancement in the growth of C7b could be attributed to the production of nutrients by C. muelleri or by the high organic material available when the microalgal cells dies. Strain C7b was found to be of no danger to the survival and performance of penaeid larvae, even at densities of up to 7.1104 CFU ml 1 in the rearing tanks (Gomez-Gil, 1998). The presence of other heterotrophic bacteria in the microalga culture did not affect the performance of strain C7b. Rico-Mora et al. (1998) found that this effect was not observed when Skeletonema costatum was cultured with V. alginolyticus and a bacterium isolated from normal microalga cultures (SK-05, probably an Aeromonas); SK-05 out-competed the vibrio, reducing its numbers 10-fold. The density of V. alginolyticus obtained by the previous authors was 6.8105 CFU ml 1 whereas in this study, the maximum density obtained was 9.6104 CFU ml 1. The higher number of V. alginolyticus found by other authors might be attributed to the species of microalga employed since the culture conditions were similar. With data presented here, it can be suggested that C. muelleri and the bacterial strain C7b can be cultured together for up to 9 days to achieve a density of 5.15106 and 6.63104 cell ml 1, respectively, and then fed to the protozoeal and mysis stages of penaeid shrimp.
Acknowledgements Carmen Bola´n Mejı´a. This work was financed by the International Foundation for Science project A/2203-2.
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