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Joseph Leopoldo Q. Laranja*, Gladys Ludevese-Pascual, Maarten Aerts, Edgar C. Amar, Peter Bossier, Peter De Schryver  
Laboratory of Aquaculture and Artemia Reference Center, Ghent University, 9000 Gent, Belgium
Aquaculture Department, Southeast Asian Fisheries Development Center, 5021 Tigbauan, Iloilo, Philippines /

It has been shown that crystalline PHB can be used as biocontrol agent in crustacean culture, while, Bacillus spp. are generally considered to be interesting probiotics for aquaculture purposes. Hence, the aim of this study was to isolate PHB accumulating Bacillus spp. from shrimp pond sediments. Subsequently, it was tested if these PHB accumulating Bacillus isolate(s) could provide beneficial effects in gnotobiotic Artemia (model system) and during culture of Penaeus monodon postlarvae.

Sediment samples from a shrimp pond in the Philippines were pasteurized to obtain a spore forming mixed bacterial culture (mBC). From this mBC, 50 pure bacterial isolates were obtained using a streak plate procedure. The 50 isolates were further analysed using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species grouping (i.e. dereplication). Nine representatives from the major MALDI-TOF-based groups were selected for PHB quantification. PHB accumulation ranged from 3- 28.6% with a total PHB yield of 0.1- 1.1 g L-1 on cell dry weight. The Bacillus isolate JL47 being the superior PHB accumulating isolate was used in in vivo experiments in gnotobiotic Artemia and in P. monodon postlarvae. In the Artemia experiment, the swimming speed and distance of the JL47-fed Artemia were significantly increased (P<0.01) as compared with those Artemia fed without JL47 addition. In P. monodon postlarvae experiment, the Bacillus sp. JL47-fed shrimp has significantly (P<0.05) higher growth (249.9 mg body weight; 3.5 cm body length) as compared with control (180.92 mg body weight; 3.1 cm body length). Survival of the shrimp was also significantly (P<0.01) higher in JL47-fed shrimp (65%) as compared with control (39%). In the challenge experiment using pathogenic Vibrio campbellii LMG 21363, survival of gnotobiotic Artemia and of P. monodon fed the Bacillus sp. JL47 was significantly  (P<0.05) higher (Artemia: 73.3%); shrimp: 52.5%) as compared with the challenge control (Artemia:10%; shrimp: 23.8%).

The study suggests a rapid procedure on how to screen and isolate PHB accumulating Bacillus spp. and also demonstrates the potential use of PHB accumulating Bacillus species as probiotics in aquaculture.

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