【作者】 陆诗敏；

【导师】 谢从新；

【作者基本信息】 华中农业大学 ， 湿地资源与环境， 2014， 博士

【摘要】 高密度集约化池塘养殖过程中产生的环境污染逐渐地引起人们的关注。大量饲料氮素残留在池塘水体和沉积物中。在这些残留氮素的生物地球化学循环过程中,氨氧化作用是硝化反应的第一步,也是限速步骤,决定了氮素循环过程的快慢。本课题以氨氧化微生物为主要研究对象,调查了四大家鱼养殖池塘中氨氧化微生物的时空分布规律,研究了空心菜浮床根系氨氧化微生物及其对净化养殖池塘水质的作用,探索了一种基于富集氨氧化微生物的生物悬浮载体池塘水质改良技术,主要研究结果如下：1)开发了一种新的沉积物微生物总DNA提取方法,琼脂糖凝胶电泳结果表明,通过此方法所获得的DNA条带整齐,大小在23kb左右。以AOA amoA基因为目的基因,通过qPCR方法对此方法进行了验证,结果表明通过本提取方法所得DNA,无论在得率还是在纯度方面均能达到土壤微生物总DNA提取试剂盒水平。2)对湖北省公安县的10口四大家鱼养殖池塘氨氧化微生物时空分布规律进行了周年调查。结果表明养殖水体中氨氧化微生物以AOB为主；AOA amoA基因在检测限以下,可能受到光抑制；未检测到Anammox菌的存在。夏季水体AOB amoA基因浓度显著高于其它季节；其它3个季节,水体中的AOB amoA基因浓度无显著差异。在沉积物中同时存在AOA、AOB和Anammox菌3种氨氧化微生物,并且都表现出季节变化规律。AOA浓度比AOB约高一个数量级,AOB最大浓度出现在秋季和冬季,AOA则出现在冬季。AOB和AOA最低浓度都出现在夏季。Anammox菌最高浓度出现在夏季和秋季,最低浓度则出现在冬季。综合分析推测DO是调控沉积物中3种氨氧化微生物的主要生态因子。统计分析表明,夏季沉积物中AOA amoA和Anammox菌16S rRNA基因以及AOB amoA和Anammox菌16SrRNA基因之间都存在显著性正相关关系(r=0.511,p <0.01; r=0.448, p<0.05; Nonparametric Correlations),表明夏季池塘沉积物中AOA和Anammox菌以及AOB和Anammox菌之间可能都存在一定的协同作用。Anammox菌16S rRNA基因浓度在夏季最高,并且和沉积物间隙水硝氮浓度之间存在显著性正相关关系(r=0.520,p<0.01; Nonparametric Correlations),表明夏季池塘沉积物中Anammox对氮素循环可能发挥着重要作用,硝氮是影响Anammox菌浓度的重要生态因子。秋季沉积物中AOB amoA基因浓度最高,并且和间隙水亚硝氮浓度之间存在显著性正相关关系(r=0.705, p<0.01; Nonparametric Correlations),表明秋季池塘沉积物中AOB对于氨氮的氧化可能起主要作用。统计分析表明冬季池塘沉积物中AOA amoA基因和Anammox菌16S rRNA基因之间存在显著性的正相关关系(r=0.794, p<0.01; Nonparametric Correlations),表明冬季池塘沉积物氮素循环过程中AOA和Anammox菌之间可能存在协同作用。3)在面积为7000m2的养殖池塘中进行了空心菜浮床净化水质实验,并研究了水体及根系氨氧化微生物的浓度及多样性。覆盖面积7.5%的空心菜浮床能够有效降低水体总氨氮浓度；同时,空心菜浮床通过调节水体pH,能够有效降低水体分子氨浓度：在晴好天气,浮床池塘分子氨浓度显著低于对照塘。空心菜根系上附着的AOBamoA基因浓度达105copy/g-106copy/g,比空心菜根系上的AOA amoA基因浓度高一个数量级左右。此外,结果显示空心菜根系上的AOB全部归属于Nitrosomonas europea lineage类群,水体和沉积物中的AOB虽然也以Nitrosomonas europea lineage类群为主,但还有32.43%的水体AOB和38.46%的沉积物AOB归属在另外两个类群：Nitrosospira ultiformis lineage和Nitrosomonas oligotropha lineage中。空心菜根系上的AOA全部归属在Group1.1b类群中,而沉积物中80%的AOA分布在Group1.1b类群中,另外20%的AOA则属于Group1.1a类群。4)研究了悬挂在池塘水体中生物悬浮载体上氨氧化微生物的富集生长状况,并对富集后的载体进行了水质净化效果研究。本研究中选择水族箱中经常用的过滤棉(PFC)作为悬浮载体,悬挂在池塘水体不同水层中,结果显示15d左右PFC上的AOB amoA基因就可达到107copy/cm3水平,然而,养殖水体AOB amoA基因浓度一直保持在10copy/cm3-102copy/cm。室内实验研究表明,富集饱和AOB后的PFC具有较高的氨氮氧化活性,当pH、温度和DO分别为7.0-8.5、28℃和6.0mg/L-7mg/L时,氨氮转化速率达0.035±0.002mg (N) cm-3(PFC) h-1。氨氮转化速率随pH(6.0-6.5；7.0-8.5；9.5-10.0)的升高而升高；氨氮转化速率和温度(14℃,21℃,28℃,35℃)之间具有极显著的正相关关系；DO (1.0mg/L-2.0mg/L;6.0mg/L-7.0mg/L)水平对氨氮转化速率没有显著性影响。PFC作为生物悬浮载体在池塘水质修复方面拥有良好的应用前景。更多还原

【Abstract】 Environmental pollution resulted from the progress of intensive aquaculture gradually attracted people’s great attention. Large numbers of nitrogen coming from the fish feed was remained in the water and sediments in aquaculture ponds. In the biogeochemical cycle of the residual nitrogen, the oxidation of ammonia (NH3) to nitrite (NO2), the first and rate-limiting step of nitrification, determines the speed of whole nitrogen cycling process. In this study, the temporal and spatial distribution of ammonia-oxidizing microorganisms were investigated in the aquaculture pond environment in which grass carp, silver carp, bighead carp and soft-shelled turtle were raised; the ammonia-oxidizing microorganisms on Ipomoea aquatica rhizoplanes and its effect on ammonium concentration in aquaculture water were studied; a water quality improvement technology was explored based on the ammonia-oxidizing microorganisms enrichment in a biological suspended carrier. The main results were as follows:1) A new total microbial DNA extraction method from sediments was developed. Results from agarose gel electrophoresis showed that the DNA obtained by this method was a single and bright DNA band in the size of23kb. The extracted DNA was also verified by qPCR with the amoA gene of AOA, and the results showed that it could rival the soil microbial total DNA Extraction Kit both in the yield and purity.2) The temporal and spatial distribution of ammonia-oxidizing microorganisms were investigated throughout the year in ten aquaculture ponds in Gong’an, Hubei. The results showed that AOB were the main ammonia-oxidizing microorganisms in water, and significantly higher copy numbers of the AOB amoA gene were observed in summer, while no significant differences were detected among the other three seasons; the concentration of AOA amoA gene was below the minimum level of detection in the freshwater column, and it was deduced that AOA was probably inhibited by light; and Anammox bacteria were not detected in the freshwater column. The AOA, AOB and Anammox bacteria were co-existed in the aquaculture pond sediments, and all of the three kinds of ammonia-oxidizing microorganisms showed typical seasonal patterns throughout the year. The concentration of the AOA amoA gene was higher than that of the AOB amoA gene in sediments by almost one order of magnitude. The maximum density of AOB was observed in autumn and winter, while the maximum density of AOA was observed in winter. The minimum densities of both AOA and AOB occurred in summer. The highest concentration of Anammox bacteria appeared in summer and autumn, while the lowest concentration of Anammox bacteria was observed in winter. DO is suggested to be the key factor determining the typical seasonal dynamics of the three kinds of ammonia-oxidizing microorganisms in sediments based on the comprehensive analysis.Anammox bacteria16S rRNA gene copy numbers showed significant positive correlation with both of AOA and AOB amoA gene copy numbers (for AOA, r=0.511,p<0.01; for AOB, r=0.448, p<0.05; Nonparametric Correlations) in sediments in summer, which indicated that a synergistic action may exist between Anammox bacteria and AOA, AOB. The highest concentration of Anammox bacteria16S rRNA gene copy numbers was observed in summer, and was significantly correlated with the NO3--N concentration in the pore water (r=0.520,p<0.01; Nonparametric Correlations), which indicated that the Anammox may play an important role in the nitrogen cycle in sediments, and NO3-is an important ecological factor regulating the Anammox. The highest concentration of AOB amoA gene copy numbers was observed in autumn, and showed significant correlation with the concentration of NO2--N in the pore water (r=0.705,p<0.01; Nonparametric Correlations), which indicated that the AOB may be responsible for the oxidation of NH4+in the pond sediments in autumn. AOA amoA gene copy numbers showed significant correlation with the Anammox bacteria16S rRNA gene (r=0.794,p<0.01; Nonparametric Correlations) in sediments in winter, which indicated that there may exist synergistic action between AOA and Anammox bacteria in winter.3) The experiment of improving aquaculture water quality by Ipomoea aquatica floating bed was conducted in the aquaculture pond (7000m2), and the abundance and diversity of the ammonia-oxidizing microorganisms on Ipomoea aquatica rhizoplanes were investigate. Results showed that the7.5%coverage rate of Ipomoea aquatica floating bed could effectively reduce the total ammonia concentration in aquaculture water; and Ipomoea aquatica floating bed could effectively reduce the concentration of unionized ammonia by adjusting the water pH. In sunny days, the concentrations of unionized ammonia in floating bed ponds were significantly lower than that of control ponds. In addition, the concentration of AOB amoA gene copy numbers of Ipomoea aquatica rhizoplanes was up to105copy/g-106copy/g, which was about10times higher than that of AOA. Results also showed that the AOB on Ipomoea aquatica rhizoplanes all belong to Nitrosomonas europea lineage, while part of the AOB in water and sediments (32.43%in water, and38.46%in sediment) belong to the other two taxa: Nitrosospira ultiformis lineage and Nitrosomonas oligotropha lineage. AOA on Ipomoea aquatica rhizoplanes all belong to Group1.1b group, while80%AOA in sediments belong to the Group1.1b, another20%AOA belong to the Group1.1a group.4) Polyethylene filter cotton (PFC) was used as a suspended biocarrier for ammonia-oxidizing microorganisms enrichment in aquaculture ponds, and the effects of temperature, pH and dissolved oxygen on the nitrification rate of enriched PFC were evaluated in the lab. The concentration of AOB amoA gene was found up to about107copy/cm3(PFC) after about a15-day incubation, while it was only101copy/cm3-102copy/cm3in the aquaculture water. The nitrification rate of the filter cotton saturated with ammonia-oxidizing bacteria reached0.035±0.002mg (N) cm-3(PFC) h-1measured at pH7.0-8.5,28℃and a dissolved oxygen concentration of6mg/L-7mg/L. The nitrification rate increased with pH (6.0-6.5;7.0-8.5;9.5-10.0), and the nitrification rate showed significant positive correlation with temperature (14℃,21℃,28℃,35℃), while the DO (1.0mg/L-2.0mg/L;6mg/L-7.0mg/L) level showed no significant effect on the nitrification rate. The results suggest that the use of filter cotton as a biological suspended biocarrier in aquaculture ponds is a feasible and cheap method to remove ammonia in situ.更多还原