凡纳滨对虾和罗氏沼虾亲虾养殖塘水质及微生物群落多样性的研究

发布时间：2018-06-11 10:32

本文选题：凡纳滨对虾 + 大棚养殖　；参考：《上海海洋大学》2017年硕士论文

【摘要】：凡纳滨对虾因具有适应能力强、生长快、营养需求低等优点已成为我国对虾养殖的主要品种之一。罗氏沼虾是一种世界性优良淡水虾种,因个体大、生长周期短、食性广、易养殖和肉质美味等优点,具有较高经济价值,是发展前景较好的特种水产品之一。2016年4-6月在上海市奉贤区潘垫村某水产养殖合作社每5天1次采集7个凡纳滨对虾大棚养殖塘的水样;实验结束时采集底泥,对水质以及池水和底泥中微生物群落多样性进行了分析,并探讨了养殖池水中微生物群落随时间的变化规律以及水质因子与微生物群落组成之间的相关性。2014年12月-2015年3月,在上海市金山区廊下镇某水产公司建立了罗氏沼虾亲虾越冬池外置式循环养殖系统。越冬池水经泵抽提至外置式生物滤器顶部,水流自顶部向下流经由纳米纤维膜构成的滤料后,再从滤器底部流出回到越冬池中,在越冬池内同时还悬挂了由普通纤维制成的人工水草。每3-4天采集水样,并在实验结束时采集水样、人工水草、纳米纤维滤料样品。对罗氏沼虾亲虾越冬池的水质进行了分析,利用高通量Miseq测序技术对池水、人工水草和纳米纤维滤料3种不同基质上的微生物群落进行了多样性分析。凡纳滨对虾大棚养殖塘水质监测结果如下:1)在凡纳滨对虾大棚养殖塘中,pH、溶解氧(DO)和温度(T)平均值分别为(8.00±0.30)、(6.76±1.41)mg·L-1和(25.9±2.91)℃,均符合凡纳滨对虾生长条件。整个养殖过程中,化学需氧量(COD)、氨氮(TAN)、总氮(TN)在各养殖塘间无显著差异(p0.05);亚硝态氮(NO2--N)、硝态氮(NO3--N)、总磷(TP)、活性磷(AP)在各养殖塘间差异极显著(p0.01)。4、5、6、7、8、12和13号塘在养殖全程中TAN平均值分别为(0.88±0.55)、(0.84±1.00)、(1.07±0.87)、(0.87±0.69)、(0.74±0.48)、(0.65±0.50)和(0.76±0.56)mg·L-1,NO2--N平均值分别为(0.24±0.16)、(0.32±0.22)、(0.44±0.17)、(0.17±0.26)、(0.28±0.24)、(0.20±0.19)和(0.18±0.21)mg·L-1,基本在凡纳滨对虾可承受范围之内。养殖过程中TP和AP含量很低,各塘平均值分别为(0.06±0.04)、(0.17±0.10)、(0.17±0.12)、(0.12±0.07)、(0.13±0.05)、(0.09±0.06)、(0.09±0.05)mg·L-1和(0.01±0.001)、(0.07±0.10)、(0.09±0.11)、(0.02±0.01)、(0.03±0.02)、(0.02±0.01)、(0.02±0.01)mg·L-1。整体来看,养殖塘水质符合凡纳滨对虾生长所需。2)罗氏沼虾亲虾越冬期间,越冬池pH维持在(7.64±0.31),DO维持在(6.01±0.31)mg·L-1,COD平均值为(4.17±2.06)mg·L-1,NO2--N平均值为(0.25±0.15)mg·L-1,TAN平均值为(0.16±0.07)mg·L-1。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40d后水质达到了相对稳定的状态。因此,通过在育苗池中悬挂人工水草,配合内含纳米纤维滤料的外置式生物滤器,可使罗氏沼虾越冬亲虾池保持良好的水质。利用高通量测序技术对凡纳滨对虾大棚养殖塘池水与底泥以及罗氏沼虾亲虾越冬池水、人工水草、纳米纤维滤料上的微生物群落结构进行研究,结果如下:1)在属水平上,凡纳滨对虾大棚养殖塘池水中共检测并鉴定出812种微生物,隶属于39个门,其中未鉴定出微生物占微生物总量的29%。以属类丰度0.1%为标准,得到85种微生物,占微生物总量的65%。在门水平上,以门类丰度0.1%为标准得到优势菌有9种,分别为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、蓝细菌(Cyanobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、绿菌门(Chlorobi)、酸杆菌门(Acidobacteria)、绿弯菌门(Chloroflexi)和芽单胞菌门(Gemmatimonadetes),其中变形菌门、放线菌门、蓝细菌和拟杆菌门占绝对优势,分别占微生物群落总丰度的60%、16%、10%和9%;厚壁菌门和绿菌门丰度相对较大,分别为3%和1%。在门类微生物中以丰度0.1%为标准,对每个塘池水中前10种微生物进行统计,在7个塘中均有优势门类5种,分别为变形菌门、放线菌门、拟杆菌门、厚壁菌门和蓝细菌;4、5和8号塘分别特有柔膜菌门(Tenericutes)、迷踪菌门(Elusimicrobia)和螺旋体门(Spirochaetes)。2)在属水平上,凡纳滨对虾大棚养殖塘底泥中共检测并鉴定出819种微生物,隶属于50个门,其中未鉴定出的微生物占微生物总量的31%。以属类丰度0.1%,得到131种微生物,占微生物总量的59%。在门水平上,以门类丰度0.1%为标准,得到16种微生物,分别为变形菌门、拟杆菌门、厚壁菌门、酸杆菌门、芽单胞菌门、放线菌门、绿菌门、柔膜菌门、硝化螺旋菌门(Nitrospirae)、丝杆菌门(Fibrobacteres)、螺旋体门(Spirochaetes)、疣微菌门(Verrucomicrobia)、蓝细菌、迷踪菌门、绿弯菌门和梭杆菌门(Fusobacteria),占微生物总量的98%;其中,变形菌门、拟杆菌门和厚壁菌门占绝对优势地位,分别占微生物总量的46%、17%和16%,酸杆菌门、芽单胞菌门、放线菌门和绿菌门占相对优势地位,分别占微生物总量的3.8%、3.1%、3%和1.3%。以门类丰度0.1%为标准,对每个塘底泥中前10种微生物进行统计,在7个塘中均有优势微生物6种,分别为变形菌门、拟杆菌门、厚壁菌门、酸杆菌门、放线菌和芽单胞菌门。3)通过物种组成热图和条形图可知:在门类水平上,在大棚养殖塘池水中,变形菌门、放线菌和拟杆菌门在养殖初期就已形成绝对优势地位,且在整个养殖过程中都处于绝对优势地位,而蓝细菌在养殖中期逐渐开始处于优势地位;在属水平上,处于优势地位的微生物会随着时间、养殖条件的改变而不断地发生变化,原先处于优势地位的微生物可能会在某一时间点处于劣势地位。整体来看,在门水平上,占优势地位的微生物对微生物群落组成贡献极大;在属水平上,微生物种类多,但丰度不大,且大部分微生物对微生物总量的都有贡献,而对这些微生物的功能作用目前尚未有了解,故不可忽略。4)利用典型相关分析(canonical correlation analysis,CCA)对凡纳滨对虾大棚养殖塘水质与水中微生物群落结构的相关性、水质与底泥中微生物群落结构的相关性进行了分析。结果发现,池水中微生物群落结构受DO、pH、COD、TN影响较大,其中p H影响最大;在养殖塘底泥中,微生物群落结构受T、ALK、AP、NO3--N影响较大,其中受T影响最大。利用高通量Miseq测序技术测定了罗氏沼虾越冬池池水、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滤料3种不同基质上的微生物群落组成。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、放线菌门、拟杆菌门、绿弯菌门、厚壁菌门、浮霉菌门(Planctomycetes)、硝化螺旋菌门、酸杆菌门和绿菌门。从分类学属的水平上对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的一个属(Comamonadaceae_unclassified),其也是3种基质的共有优势菌属;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。
[Abstract]:Penaeus vannamei has become one of the main varieties of prawns in China because of its strong adaptability, fast growth and low nutrition demand. One of the species of aquatic products in 4-6 months of.2016 in Fengxian District, Shanghai, an aquaculture co - operative of Pan mat village, Fengxian District, collected water samples for 1 times every 5 days, and the sediment was collected at the end of the experiment. The microbial community diversity in water and pond water and sediment was analyzed, and the microbial community in the aquaculture pool was discussed with time. The correlation between the changes of the water quality and the composition of the microbial community in the December -2015 year of December in.2014, Jinshan District, Shanghai City, an aquaculture system was set up in the Aquatic company of the lobster prawns in Jinshan District, Shanghai City, which was pumped to the top of the external Biofilter by pumping the water through the pump. After the filter material made up of the rice fiber membrane, the water samples made from ordinary fiber were suspended from the bottom of the filter back to the overwintering pool, and the water samples were collected every 3-4 days, and the water samples were collected at the end of the experiment, the artificial water and grass and the nanofiber filter samples were collected. High throughput Miseq sequencing technology was used to analyze the diversity of microbial communities on 3 different substrates of pool water, artificial water grass and nanofiber filter material. The results of water quality monitoring of shrimp pond culture ponds in Van Nanbin were as follows: 1) the average values of pH, dissolved oxygen (DO) and temperature (T) were (8 + 0.30) and (6.76 + 1.41) mg respectively in the pond culture ponds of Penaeus vannamei. L-1 and (25.9 + 2.91) C were all suitable for the growth conditions of Penaeus vannamei. In the whole culture process, the chemical oxygen demand (COD), ammonia nitrogen (TAN) and total nitrogen (TN) had no significant difference (P0.05), and nitrite nitrogen (NO2--N), nitrate nitrogen (NO3--N), total phosphorus (TP), and active phosphorus (AP) were very significant (P0.01).4,5,6,7,8,12 and No. 13 ponds in the pond. The average values of TAN were (0.88 + 0.55), (0.84 + 1), (1.07 + 0.87), (0.87 + 0.69), (0.74 + 0.48), (0.65 + 0.50) and (0.76 + 0.84) mg. L-1 respectively. The mean value of NO2--N Within the breeding process, the content of TP and AP is very low, and the average values of the ponds are (0.06 + 0.04), (0.17 + 0.10), (0.17 + 0.12), (0.13 + 0.05), (0.09 + 0.06), (0.09 + 0.12), (0.12), (0.12), (0.12), Mg. During the winter of the growth of Penaeus vannamei.2), during the winter of the shrimp, the pH of the wintering shrimp was maintained at (7.64 + 0.31), DO was maintained at (6.01 + 0.31) mg. L-1, the average value of COD was (4.17 + 2.06) mg. L-1, NO2--N average value was (0.25 + 0.15) mg L-1, and TAN average was (0.16 + 0.07). The water quality reached a relatively stable state after the opening of the circulating water system for about 40d. Therefore, by hanging artificial water grass in the nursery and combining with the external biofilter containing nanofiber filter material, the shrimp pond in the winter can keep good water quality. The microbial community structure on the wintering pond water, artificial water and grass and nanofiber filter material were studied. The results were as follows: 1) at the level of the genus, 812 kinds of microbes were detected and identified in the pond water of Penaeus vannamei aquaculture, which belonged to 39 doors, of which 29%. was not identified as the total amount of microbes. Degree 0.1% was the standard to obtain 85 kinds of microbes, which accounted for 65%. of total microbial biomass at the gate level, and 9 species of dominant bacteria were obtained by the standard of class abundance 0.1%, which were deformable bacteria gate (Proteobacteria), actinomycetes (Actinobacteria), cyanobacteria (Cyanobacteria), pseudomycetes (Bacteroidetes), Firmicutes, Chlorobi, acid. Bacilli (Acidobacteria), Chloroflexi and Gemmatimonadetes, including deformable bacteria, actinomycetes, cyanobacteria and Pseudomonas, accounted for 60%, 16%, 10% and 9% of the total abundances of microbial communities, and the abundance of the phylum and phylum was 3% and 1%., respectively, in the phylum species. Degree 0.1% is the standard. There are 10 kinds of microorganisms in each pond water. There are 5 dominant species in 7 ponds, which are deformable bacteria doors, actinomycetes, bacteriobacteria, thick wall bacteria and cyanobacteria; 4,5 and No. 8 are Tenericutes, Elusimicrobia and Spirochaetes.2) at the level of the genus 819 kinds of microbes were detected and identified 819 kinds of microbes in the pond sediment of Penaeus vannamei, which belonged to 50 doors, of which the unidentified microbes accounted for 31%. of the total amount of microbe, 0.1% of the genera, 131 kinds of microbes, which accounted for the total amount of 59%. at the gate level, and 16 kinds of microbes were obtained by the standard of the phylum abundances 0.1%. Bacteria gate, Pseudomonas, bacilli, acid bacilli, bud monomonas, actinomycetes, actinomycetes, green bacteria gate, soft membrane bacteria gate, Nitrospirae, Fibrobacteres, Spirochaetes, Verrucomicrobia, cyanobacteria, trace bacteria, phylum aeruginosa, and Clostridium (Fusobacteria), accounting for microbes. 98% of the amount, among them, deformable bacteria doors, Pseudomonas and thick walled bacteria accounted for 46%, 17% and 16% of the total microbial total, acid bacilli, buds, actinomyces, actinomycetes and green bacteria, accounting for 3.8%, 3.1%, 3% and 1.3%., respectively, and 0.1% as the standard, and 10 in the bottom of each pond. There are 6 dominant microbes in the 7 ponds, including deformable bacteria gate, Pseudomonas, actinomycetes, acid bacilli, actinomycetes and bufominomonas.3 through species composition heat map and bar chart. It is known that in the class level, in the pond pond water, deformable bacteria gate, actinomycetes and Pseudomonas aeruginosa at the early stage of culture. It has formed an absolute dominant position and is in an absolute dominant position during the whole breeding process, and the cyanobacteria gradually begin to be in the dominant position in the medium period of culture. At the level, the dominant microbes will change continuously with the time and the culture conditions, and the previously dominant microorganisms may be able to change. As a whole, the dominant microorganism contributes greatly to the composition of microbial communities on the level of the door; at the level of the genus, there are many kinds of microbes but not much abundance, and most microorganisms contribute to the total amount of microbes, and the function of these microbes has not yet been understood. The correlation between water quality and microbial community structure in aquaculture ponds of Penaeus vannamei was studied by.4 (canonical correlation analysis, CCA). The correlation between water quality and microbial community structure in sediment was analyzed. The results showed that the microbial community structure in the pool was greatly influenced by DO, pH, COD, and TN. The influence of medium P H was the greatest. In the sediment of culture pond, the microbial community structure was greatly influenced by T, ALK, AP and NO3--N, among which T was most affected. The high throughput Miseq sequencing technology was used to determine the wintering pool water of the Macrobrachium Roche, the artificial water and grass (ordinary fiber membrane) in the pool and the nanofiber filter material of the external biofilter on 3 different substrates. Microbial composition and diversity on different substrates were different. 64 species of bacteria were detected and identified on 3 substrates, belonging to 9 families, 64 genera, including deformable fungi, actinomycetes, Pseudomonas, green Bens, Planctomycetes, nitrifying spiraces, acid bacilli and green bacteria. At the level of the genus, the bacteria on the 3 substrates were analyzed. It was found that the highest content of the aquaculture pool was Comamonadaceae_unclassified, which was also the dominant genus of 3 substrates; the ordinary fiber membrane was Inhella, and the nanofiber membrane was the bacterial community on the Fusibacter.3 substrate. The order of diversity is nanofiber membrane, common fiber membrane, aquaculture pond water.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S966.12;S968.22

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