一株兼具氨氮去除能力和对副溶血弧菌拮抗作用的有益菌的筛选及其初步应用

Identification and Application of a Probiotic Strain Functioning in Both Ammonia Nitrogen Removal and Vibrio parahaemolyticus Antagonism

中文关键词: 脱氮菌  副溶血弧菌  杀鱼假交替单胞菌  拮抗作用

英文关键词: Denitrifying bacteria  Vibrio parahaemolyticus  Pseudoalteromonas piscicida  Antagonism

中文摘要:

      采用无机氮筛选培养基，从对虾养殖水体和对虾肠道中分离到3株具有脱氮效果的异养硝化菌株(编号分别为20131023A00、20131023A01和20131023A05)，它们在氨氮含量为0.12%的液体筛选培养基上(pH=7.2)，28℃经24 h对氨氮的转化能力分别为(38.9±0.1)%、(43.1±0.4)%和(49.9±0.5)%；采用纸片法拮抗实验选出菌株20131023A05对副溶血弧菌(Vibrio parahaemolyticus)具有拮抗作用，在1.57×105 CFU/cm2、1.57×104 CFU/cm2和1.57×103 CFU/cm2的副溶血弧菌平板上产生的抑菌圈直径分别为(9.14±0.05) mm、(11.57±0.03) mm和(13.59±0.02) mm。经16S rDNA序列测定，表明该菌株与杀鱼假交替单胞菌(Pseudoalteromonas piscicida)有最近的亲缘关系。在日本囊对虾(Marsupenaeus japonicus)养殖水体中加入该株菌至2.5×105 CFU/ml时，该菌株表现出对副溶血弧菌注射感染的保护作用，该菌株浸浴组的日本囊对虾相对存活率(RPS)为35%。在凡纳滨对虾养殖水体中每3 d加入该株细菌(3.13×104 CFU/ml)，每天加入70%饲料量的赤砂糖，经60 d养殖，水体中氨氮含量比对照组及其他组显著降低。菌株20131023A05兼具去除氨氮和对副溶血弧菌拮抗作用，在对虾养殖生产中有广阔的应用前景。

英文摘要:

      Three bacterial strains, 20131023A00, 20131023A01, and 20131023A05, were isolated from shrimp guts and the farming ponds using inorganic selecting medium. When cultured in the liquid selecting medium (pH=7.2) containing 0.12% ammonia nitrogen at 28℃ for 24 h, the ammonia conversion rates of the three strains were (38.9±0.1)%, (43.1±0.4)%, and (49.9±0.5)% respectively. Using the paper disk method, we identified that 20131023A05 was the only strain that antagonized Vibrio parahaemolyticus. We measured the diameters of inhibition zones produced by Strain 20131023A05 on the plates coated with V. parahaemolyticus at the densities of 1.57×105 CFU/cm2, 1.57×104 CFU/cm2, and 1.57×103 CFU/cm2, and the diameters were (9.14±0.05) mm, (11.57±0.03) mm, and (13.59±0.02) mm respectively. It was identified that the strain had the closest genetic relationship with Pseudoalteromonas piscicida according to 16S rDNA sequencing. Marsupenaeus japonicus immersed with Strain 20131023A05 at 2.5×105 CFU/ml showed resistance to intramuscular-injected V. parahaemolyticus and the relative survival rate (RPS) was 35%. We applied strain 20131023A05 at 3.13×104 CFU/ml once every three days as well as brown sugar (70% of diet) every day in the culture water of Litopenaeus vannamei for 60 days. Subsequently the concentration of ammonia nitrogen in the water became significantly lower than the control and other groups. Our results suggested that Strain 20131023A05 could function in both ammonia removal and V. parahaemolyticus inhibition, therefore may have great potentials in shrimp farming industry.