A Primary Study On Isolation, Screening And Removal Characteristics Of Probiotics In Ponds Of Shrimp And Crab

In the early experiment, the sediment was collected from culture ponds for Portunus trituberculatus and Marsupenaeus japonicus in Ganyu County, Lianyungang City, Jiangsu Province. And the compound bacteria, collected from the sediment, was enriched and domesticated for five or six cycles in specific culture mediums, when every cycle finished, the COD and ammonia-nitrogen removal characteristics of the compound bacteria were tested, respectively. At the end of all cycles, bacterial strains were isolated from the compound bacteria, and through 16S rRNA sequences analysis, blood plates and the removal characteristics of each strains, they were identified and preliminarily screened. Then, the screened bacterial strains were combined in propotion. and COD and ammonia-nitrogen removal characteristics of the combined strains were tested, respectively. At the end of experiment, the effects of several different environmental factors on inorganic nitrogen removal of Bacillus pumilus (strain E6) were examined in the laboratory. The experimental design and significant results were listed below:1. A primary study on enrichment, domestication, isolation and screening of organic-pollutant-degrading bacteria in ponds of shrimp and crabThe compound bacteria collected from the sediment of culture ponds for shrimp and crab was enriched and domesticated for six cycles (7 days per cycle) by adding shrimp feed to specific culture medium regularly. When the fourth, fifth or sixth cycle finished, in feed solution, where the content of organic pollutants (COD) was about 1000mg/L and the inoculation quantity was controlled in 10%, the highest COD removal rate of 64%,70%, or 88% of the bacteria within 5 days was recorded respectively. At the end of all cycles,16 bacterial strains were isolated from the compound bacteria, and according to 16S rRNA sequence similarity analysis,8 of them were identified as vibrio sp.,1 as Bacillales sp.,2 as Bacillus sp.,2 as Pseudoalteromonas sp.,1 as Aeromonas sp. and 1 as Shewanella sp.. After preliminarily screening by blood plates,4 bacterial strains were selected:1 was Bacillales sp.,1 was Bacillus sp. and 2 were Pseudoalteromonas sp.. In feed solution, with an inoculation of 1.0×107cfu/mL. the 4 bacteria were able to degrade the feed and the highest COD removal rate of 40%,40%,56% and 46% within 5 days was recorded respectively. When the 4 bacterial strains were combined in propotion, the highest COD removal rate of 72% within 5 days was recorded. Therefore, it can be concluded that both of the compound bacteria after enrichment and domestication and the 4 bacteria after isolation and screening in this study have potential application in pollution control for culture ponds of shrimp and crab.2. A primary study on enrichment, domestication, isolation and screening of ammonia-nitrogen-degrading bacteria in ponds of shrimp and crabThe compound bacteria, which could degrade ammonia-nitrogen and was collected from the sediment of culture ponds for shrimp and crab, was enriched and domesticated for five cycles (14 days per cycle) in specific culture medium, when every cycle finished, in specific degrading solution, where the initial concentration of ammonia-nitrogen was about 120mg/L and the inoculation quantity was controlled in 10%, the highest ammonia-nitrogen removal rate of 29%, 60%,69%,74%,72% of the bacteria within 5 days was recorded respectively. After five cycles, 12 bacterial strains were isolated from the highly effective compound bacteria, on the basis of 16S rRNA sequence similarity analysis,8 of them were identified as vibrio and 4 as Halomonas. After preliminarily screening by blood plates,6 bacterial strains were selected:2 were Bacillus and 4 were Pseudoalteromonas. The highest ammonia-nitrogen removal rate within 5 days of the 6 bacteria were 59%,43%,33%,42%,41%,40% respectively with an inoculation of 1.0× 106cfu/mL in solution, and the highest removal rate of the 6 bacteria were 69%,55%,71%, 88%,79%,51% respectively with an inoculation of 1.0×107cfu/mL in solution. When the 6 bacterial strains were combined in propotion, combined strains C has the highest ammonia-nitrogen removal rate within 5 days, and it was 62%.3. Removal characteristics of a heterotrophic nitrifying-aerobic denitrifying bacteria strain E6In this study, the effects of several environmental factors, including time, temperature, salinity, pH, quantity of inoculated bacteria. C:N ratio, and carbon sourceon on NH4+-N, NO2--N. NO3--N and total inorganic nitrogen removal of a potential probiotic bacteria strain E6, which was isolated from the sediment of shrimp and crab ponds, were examined in the laboratory. The experimental results indicated that, when strain E6 was inoculated into nitrogen degrading solution, where the initial concentration of NH4+-N was about 107.72 mg/L. NO2--N was about 8.55 mg/L. NO3--N was about 39.20 mg/L, the removal rate of NH4+-N. NO2--N, NO3--N and total inorganic nitrogen reached 47.59%、93.22%、64.84% and 54.45% within 5 days; the optimal conditions for strain E6 to have better inorganic nitrogen removal performance were showed as follows:the temperature was 28-34℃, salinity was 20-60%o, pH was (6-7), the quantity of bacteria inoculated was 5%, the C:N ratio was 20, and the carbon source was sucrose; the highest removal rate of NH4+-N, NO2--N, NO3--N and total inorganic nitrogen reached 70%,100%,81% and 75% respectively. Therefore, it can be concluded that Bacillus pumilus (strain E6) is a valuable bacterium with ability of simultaneous heterotrophic nitrification and aerobic denitrification, and shows great application potential in mariculture for shrimp and crab.