罗氏沼虾亲虾越冬池中不同基质上微生物群落多样性分析

Microbial community diversity on different substrates in Macrobrachium rosenbergii over-wintering cultivation

摘要:

【目的】了解罗氏沼虾亲虾越冬时循环养殖系统对水质的调控效果，探明其中微生物群落的作用。【方法】采集循环养殖系统运行88 d后的越冬池池水、池中人工水草(普通纤维膜)以及外置式生物滤器中的纳米纤维膜等3种不同基质上的微生物，利用DNA抽提、PCR扩增和定量以及高通量Miseq测序技术等对3种不同基质上的微生物进行16S rRNA基因序列V4?V5区的测定和分析，并根据测序得到的双端测序读长(Pair-end reads)进行质量控制和过滤，之后进行操作分类单元(OTU)聚类分析，并基于OTU对微生物群落的多样性指数和群落结构进行分析；每3?4 d对越冬池池水水质进行监测。【结果】养殖池塘的水质保持在良好的状态，其中氨氮和亚硝氮浓度控制在0.17±0.08 mg/L和0.28±0.15 mg/L；不同基质上的微生物组成和多样性都不相同。在3种基质上共检测并鉴定出细菌64种，隶属于9门64属，包括变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、绿弯菌门(Chloroflexi)、厚壁菌门(Firmicutes)、浮霉菌门(Planctomycetes)、硝化螺旋菌门(Nitrospirae)、酸杆菌门(Acidobacteria)和绿菌门(Chlorobi)。从属水平上对3种基质上的细菌进行分析，发现养殖池水中含量最高的为丛毛单菌科下的一个未分类类群(Comamonadaceae_unclassified)，其也是3种基质的共有优势类群；普通纤维膜上为Inhella，纳米纤维膜上则是小纺锤状菌属(Fusibacter)。3种基质上细菌群落多样性顺序为：纳米纤维膜>普通纤维膜>养殖池水。通过对亲虾越冬养殖全过程的水质监测，发现越冬期间亲虾池水质始终保持在良好状态，并且在循环水系统开启约40 d后水质达到了相对稳定的状态。【结论】通过在育苗池中悬挂人工水草，配合内含纳米纤维膜的外置式生物滤器，可使罗氏沼虾越冬亲虾池保持良好的水质。随着新型材料科学的发展，开发出适用于水产养殖业的滤料很有必要。

Abstract:

[Objective] The aim of this study was to check the effect of water quality control by RAS (Recirculating Aquaculture System) and analyze the function of microbial community on different substrates during the period of Microbrachium rosenbergii over-wintering cultivation. [Methods] The microbial samples were collected from pond water, artificial aquatic plants which were composed of normal fiber membrane and nano fiber membrane in external biofilter after RAS being operated for 88 days. The V4 and V5 regions of 16S rRNA gene on three different substrates microbial were analyzed using DNA extraction, PCR amplification and quantification, high-throughput miseq sequencing technology. Sequence data was processed by read trimming and identification of V4?V5 sequences, followed by filtering and assigning the operation taxonomic units (OTU). Based on the OTU analyzed the microbial community diversity index and structure. Water quality was monitored every 3?4 days with the national standard method. [Results] The pond water quality maintained in a good condition that ammonia-nitrogen and nitrite-nitrogen kept at 0.17±0.08 mg/L and 0.28±0.15 mg/L, respectively. Bacteria composition and community diversity varied in three substrates and 64 species were identified which belonged to 64 genus and 9 phylum including Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, Nitrospirae, Acidobacteria and Chlorobi. On the level of genus, Comamonadaceae_unclassified was the most dominant genus in pond water which both appeared in the other two substrates. Inhella dominated in surface of normal fiber while Fusibacter was predominant in nano fiber membrane surface. Nano fiber membrane had the highest bacteria community diversity, followed by normal fiber membrane and then pond water. Water quality of over-wintering cultivation maintained at a relatively steady state after operating recirculating aquaculture system for 40 days. [Conclusion] It is viable to regulate and control water quality of over-wintering cultivation by using fiber membrane as a microbial substrate. With the development of new material science, it is necessary to develop a filter material suitable for aquaculture.