两种斑节对虾养殖模式水质净化效果与细菌群落结构的比较分析

Comparison of Water Quality Purification and Bacterial Community Structure Between Two Culture Modes of Tiger Shrimp Penaeus monodon

为了提高对虾养殖系统水质净化能力,改善对虾养殖水环境,利用红糖+枯草芽孢杆菌和聚氨酯填料+硝化细菌分别构建生物絮团和内循环斑节对虾(Penaeus monodon)养殖系统,比较两个系统在对虾标苗和养殖阶段的水质净化效果和细菌群落结构,从微生物学层面探究其水质净化机理。将体长(0.5±0.1)cm的5日龄仔虾养殖在1 m^(3)水体的帆布池中,标苗期密度为5 000 ind.·m^(-3),养殖期降为400 ind.·m^(-3)。标苗结束时,各帆布池底铺约3 cm厚的砂子。生物絮团系统中加活化后的枯草芽孢杆菌,每天一次性加50%日投饲量的红糖,每隔7 d加0.5 L活化后的芽孢杆菌,每隔14 d换水20%。内循环系统中悬挂已挂膜的聚氨酯填料包,其上表面浸没在水中,内部放一个气石。每个系统设2组平行,各阶段养殖周期分别为20 d和40 d。实验结束时,采集生物絮团系统水样、底砂和内循环系统生物膜、水样及底砂的细菌样品,提取DNA,利用Illumina Mi Seq平台对DNA样品进行高通量测序。结果显示,生物絮团系统氨氮和亚硝酸盐氮浓度波动变化较大,显著高于内循环系统(P<0.05);加芽孢杆菌和换水后,氨氮和亚硝酸盐氮浓度有所下降。生物絮团系统硝酸盐氮浓度显著低于内循环系统(P<0.05);化学需氧量显著高于后者(P<0.05)。两个系统总磷浓度无显著差异(P>0.05)。细菌群落结构分析表明,在44个菌门中,有25个为所有样品所共有,5个样品优势菌门均为变形菌门(Proteobacteria)和拟杆菌门(Bacteroidota),其在生物絮团系统水样、底砂和内循环系统生物膜、水样及底砂中的相对丰度之和依次为58.51%、55.44%、61.48%、73.42%及65.69%。生物絮团和内循环系统中分别存在芽孢杆菌属(Bacillus)、栖砂杆菌属(Arenibacter)、珞珈苍黄色杆菌属(Luteolibacter)等和硝化螺旋菌属(Nitrospira)、硝化刺菌属(Nitrospina)、假交替单胞菌属(Pseudoalteromonas)等具有有机物降解、水质净化及对虾益生作用的菌属。由此可知,生物絮团系统对氨氮和亚硝酸盐氮的净化能力不如内循环系统;生物絮团和内循环系统门和属水平上细菌群落组成及其相对丰度均存在差异。

In order to improve the water quality purification capacity in a shrimp culture system and to improve the water environment of shrimp culture,the effects of water quality purification and bacterial community structure were comparatively investigated in two systems,one prepared by brown sugar+Bacillus subtilis(biofloc system)and the other polyurethane packing+nitrifying bacteria(internal circulation system),in the intermediate rearing and culture stage of tiger shrimp Penaeus monodon by high-throughput sequencing to probe into the mechanism of water quality purification from the level of microbiology.The 5-day-old tiger shrimps with body length of(0.5±0.1)cm were cultured in a canvas pond of 1 m^(3) water body at stocking density of 5000 ind.·m^(-3) in the intermediate rearing period for 20 d,and at stocking density of 400 ind.·m^(-3) in the culture period for 40 d.At the end of the intermediate rearing,the bottom of each canvas pond was spread with sand about 3 cm thick.In the biofloc system,0.5 L of activated Bacillus subtilis was added weekly and brown sugar was daily added at dose of 50%of the daily feeding amount,with 20%of the water was changed biweekly.In the internal circulation system,a polyurethane packing package with a hanging film and a gas stone is placed inside was suspended,and the upper surface was immersed in water.Each system was set with 2 parallel groups.At the end of the experiment,the bacterial samples of water,bottom sand in the biofloc system and the bacterial samples of biofilms,water and bottom sand in the internal circulation system were collected for DNA extraction,and high-throughput sequencing of DNA using the Illumina MiSeq platform.The results showed that there were significantly higher concentrations and greater fluctuation of ammonia nitrogen and nitrite nitrogen in the biofloc system than those in the internal circulation system(P<0.05).The concentrations of ammonia nitrogen and nitrite nitrogen were shown to be declined in the biofloc system after activated Bacillus subtilis was added and the water was changed.The concentration of nitrate nitrogen was significantly lower and the concentration of chemical oxygen demand was significantly higher in the biofloc system than those in the internal circulation system(P<0.05),without significant difference in total phosphorus concentration between the two systems(P>0.05).The analysis of bacterial community structure showed that 25 out of 44 bacterial phyla were shared by all samples,with the dominant bacterial phyla of Proteobacteria and Bacteroidota in the 5 samples,and the relative abundance sum of 58.51%in water in the biofloc system,55.44%in bottom sand in the biofloc system,61.48%in biofilms in the internal circulation system,73.42%in water in the internal circulation system and 65.69%in bottom sand in the internal circulation system.Bacillus,Arenibacter and Luteolibacter were identified in the biofloc system and Nitrospira,Nitrospina and Pseudoalteromonas were identified in the internal circulation system.These bacteria degraded organic matters,purified water quality and promoted the growth of tiger shrimp.The findings indicated that the purification capacity of biofloc system for ammonia nitrogen and nitrite nitrogen was not as good as that of internal circulation system.There were differences in the composition and relative abundance of bacterial community between the biofloc and internal circulation systems at the level of phylum and genus.