Study On The Ammonia-Oxidizing Microorganisms In The Freshwater Aquaculture Pond Environment

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.