| The accumulation of ammonia nitrogen and nitrite nitrogen and the organic pollution is the main inducement factor of aquaculture water deterioration, diiseases occurrence and quality decreasing with loach(Misgurnus anguillicaudatus). The use of beneficial microorganisms to the bioremediation is the hot spot of research and development at present. To screen and gain dominant strains which have excellent water purification effects and preparation performance is the key to develop efficient micro ecological preparation. At present, a lot of probiotics has been used in aquaculture, but screening, growth characteristics and application research for the use of probiotics on loach is rarely reported. In this study, One probiotic Bacillus subtilis NQ1 was isolated and purified from the aquaculture water of loach.The growth characteristics, aquaculture characteristics, security, the purification ability of water quality and the effects on immunity of loach were studied.The result could provide the basis for the popularization and application of the loach breeding.The main results were as follows:1. The screening of probiotic bacillus from the aquaculture water of loach.4 Bacillus strains were isolated and purified from the aquaculture water. One dominant strain,Bacillus NQ1,was obtained through the degradation of ammonia and nitrite nitrogen screening tests.The two days ammonia degradation rate of NQ1 was33.5% and nitrite nitrogen degradation rate was 99.2%,when the initial ammonia and nitrate nitrogen concentratuion was 5mg/L and 2mg/L in simulated fresh aquaculture water. Based on morphological features and results of physiological and biochemical characteristics, NQ1 was identified as Bacillus subtilis according to The Manual of Systematic Methods of Determinative Bacterial and Bergey’s Manual of Deteminative Bacteriology. Study on the growth characteristics and it showed that, NQ1 could enter the logarithmic growth phase after 6h and achieve the stable growth stage after 16 h. At the same time, it has better adaptability to temperature, salinity, pH.The results of safetytests showed that the strain NQ1 of test concentration(1×107 cfu/mL) was safe on loach.Besides, mixed the 1×107cfu/mL of NQ1 and 1×107cfu/mL of Aeromonas hydrophila on aquaculture water, the blank group was used as the negative control group, the Aeromonas hydrophila group was used as the positive control group, fostered the loach14 days. The results indicated, the survival rate of loach of NQ1 and Aeromonas hydrophila compound group was higher than the positive control group by 25%.2. The growth characteristics and culture medium optimization of Bacillus subtilis NQ1LB Medium was used as culture for cell multiplication of NQ1. The effects of culture time, initial pH, temperature, salinity and rotation speed were studied. The results showed: the optimal time, initial pH, temperature, salinity, volume of liquid medium and inoculation volume were 20 h, 7.4, 28 ?C, 0.5%, 180 r/min, 80mL/250 mL and 10% respectively. Compositions of culture media were optimized through single factor test and orthogonal experiment of L9(34). The results were as follows: peptone(2%), glucose(2%), NaCl(0.3%), and under those optimal conditions, the number of NQ1 reached 10.44×108 CFU/ml.3. The effect of Bacillus subtilis NQ1 on aquaculture water quality of loachWe added Bacillus subtilis NQ1 at the concentration of 0, 1×105, 1×106 and1×107cfu/mL to the aquaculture water, and studied the effect of NQ1 on water quality indicators of loach. The results showed that, after using NQ1 for 14 days, ammonia degradation rate(52.67%), nitrite degradation rate(50.18%) and sulfide degradation rate(48%) at concentration 1×107 cfu/mL were the highest value in three experimental groups, and pH(7.76 ~ 7.65) fluctuation of it is smallest. Thereby indicating, water quality of loach farming pond was obviously purified by 1×107 cfu/mL of Bacillus subtilis NQ1.4. The effect of Bacillus subtilis NQ1 on digestive enzymatic activity and nonspecific immunity of loachWe added Bacillus subtilis NQ1 at the concentration of 0(comparison), 1×105(low concentration), 1×106(medium concentration) and 1×107cfu/mL(high concentration) to the aquaculture water, and studied the effect of NQ1 on digestive enzymatic activity and nonspecific immunity of loach. The results showed that, after using NQ1 for 14 days,compared with group of comparison, the activity of intestinal amylase(AMS) and intestinal lipase(LPS) were evidently enhanced at medium concentration and highconcentration(P<0.05), it was better in high concentration group than that in medium concentration group, but the difference was not evident(P>0.05), low concentration group does not evidently effect the activity of AMS and LPS in intestines of loach(P>0.05), it showed that Bacterial strains preferably improved intestinal digestive enzyme activity of loach. At the same time, compared with group of comparison, the numbers of red blood cell(RBC), white blood cell(WBC), phagocytic percentage(PP)of leukocyte in blood, phagocytic index(PI),and activity of lysozyme(LSZ) in liver,acid phosphatase(ACP), alkaline phosphatase(AKP), superoxide dismutase(SOD),peroxidase(POD) and total antioxidant capacity(T-AOC) were evidently higher in the high concentration group(P <0.05), and activity of the glutamic oxalocetie transaminase(GOT) in liver and glutamic pyruvic transaminase(GPT) was not evidently affected(P>0.05). Overall, the effect in the high concentration is better than that in medium concentration. Therefore, adding Bacillus subtilis NQ1 at the concentration of1×107cfu/mL to water will evidently improve digestive enzymatic activity and nonspecific immunity of farming loach.In summary, Bacillus subtilis NQ1 is potential to apply and develop as probiotics. |
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