The Use of Purple Nonsulfur Photosynthetic Bacteria to Maintain Water Quality, Sources of Single Cell Protein and Bioactive Compounds for Shrimp Cultivation

Abstract

Purple nonsulfur bacteria (PNSB) as beneficial bacteria were isolated from various shrimp ponds in Southern Thailand for promising strains selection with the purpose to use them in shrimp cultivation. Among 185 isolated PNSB were screened based on probiotics properties in vitro; there were only 4 strains (SS15, S3W10, TKW17 and STW181) showed strong activities for producing digestive enzymes, vitamin B12 and antivibrio compounds. These selected PNSB were identified as Rhodobacter sphaeroides for strains SS15, S3W10 and TKW17 and Afifella marina for strain STW181. They were proved for their probiotic properties in vivo as a mixed culture (each at 1 × 108 cells mL ̄1) for 3 sets (T1: S3W10+SS15, T2: S3W10+TKW17 and T3: S3W10+STW181), by inoculating into rearing water of postlarval Litopenaeus vannamei every week (1-7) for 60 days; and then challenge white shrimp with acute hepatopancreatic necrosis disease (AHPND) causing V. parahaemolyticus SR2 at 4 x 104 cells mL1 by continuing cultivation for 10 days. All PNSB sets significantly enhanced the digestive enzyme activities and shrimp growth with their proliferation in shrimp gastrointestinal tract although the shrimp survival was not significantly different. However, the cumulative mortality of shrimp exposed to a virulent AHPND - V. parahaemolyticus SR2 was a remarkable decrease in all PNSB sets as 40% in control to 21, 24 and 32% in T1, T3 and T2 sets. The probiotic PNSB were also investigated for their ability to control water quality using a mixed culture at a ratio of 1: 1 with the same protocol as the previous experiment. Throughout 8 weeks of white shrimp cultivation, the effective sets that reduced NH4+ and also promoted shrimp growth were T1 and T3. Hence, PNSB strains (SS15, S3W10 and STW181) were used as a mixed culture for each at 1 × 108 cells mL1 by adding into rearing water for assessment their potential to prevent AHPND as all shrimp were challenged at day 15 with AHPND - V. para- haemolyticus SR2 (1 × 105 cells mL1) during shrimp cultivation for 30 days. Inoculated PNSB sets (positive control: only PNSB inoculation, and treatment: both PNSB and SR2 inoculations) produced a better water quality than that found in native control (no inoculation) and challenge test (only SR2 inoculation). This experiment confirmed their probiotic properties as they colonized in intestinal shrimp tract, and accompanying to control water quality for enhancement shrimp growth and increase 11% survival rate of challenged shrimp with AHPND strain SR2. Conditions for producing antivibrio compounds of probiotic PNSB (SS15, TKW17 and STW181) were investigated for obtaining optimal conditions and found that their optimum conditions were close to the conditions of shrimp cultivation. These antivibrio compounds mainly consisted of protein, lipid, and carbohydrate; and they were stable under wide variations of pH and temperature that was suitable for shrimp cultivation. The antivibrio compounds acted as bactericidal action as the evidence of damaged cells with many holes and also showed bacteriolytic activity. One of the purified antivibrio compounds from strain SS15 was characterized and found that it was a low molecular weight (<3000 Da) with weak cationic compound containing - NH2 group. In addition of using PNSB as probiotics they were also investigated for the potential to be used as single cell protein (SCP), and strains SS15 and STW181 were selected as SCP on the basis of protein, photopigments and essential amino acids contents. A mixture of both lyophilized cells (1:1) at 1, 3 and 5% (w/w) were mixed well with commercial shrimp feed to obtain modified shrimp feed recipes; Diet 1, Diet 2 and Diet 3, respectively for feeding white shrimp starting from postlarval until early juvenile for 60 days. Levels of NH4+, NO2, NO3 ̄ and COD in rearing water from Diet 2 and 3 sets were significantly higher than control set; however, Diet 1 set showed the lowest levels of these water parameters among modified Diet sets and closed to a control set. Based on shrimp growth performance, Diet 1 set was better than other modified diet sets including control set. The maximum shrimp survival as 85% was also observed in Diet 1 set although no significant difference with other sets as in the control set was 80%. Moreover, Shrimp in all modified shrimp diet sets showed increasing of immune responses (total hemocyte count, and activities of phenoloxidase and superoxide dismutase) and hepatopancreas histopathology of shrimp in a good condition as healthy shrimp. Overall this thesis proved that the four selected PNSB strains as beneficial microbes have the great potential to be used in white shrimp cultivation.