为了探讨农用灌溉池塘蓄积的生活污水在富氧条件下反硝化细菌丰度变化及其与水质参数的相关性，应用实时荧光定量聚合酶链式反应技术对富氧灌溉池塘水体不同深度水层中（上层10～30 cm、中层90～110 cm、和下层150～170 cm）反硝化功能基因丰度昼夜变化特征进行了研究。结果表明：水质参数中溶解氧(dissolved oxygen，DO)浓度、pH值、水温、光照度及NO3--N浓度均具有垂向分层效应，随水层的加深而逐渐降低，NH4+-N及PO43--P浓度则呈现与前者相反的趋势，随水层加深而逐渐升高；nirK，nirS和nosZ 3类反硝化基因受DO浓度和pH值调节作用显著，均表现为白天丰度高，晚间丰度低的时间效应；3类反硝化基因丰度整体表现为表层水体中的基因丰度高，其次为中层水体，底层水体中的丰度最低的趋势，即同样表现出垂向分层效应。nirS基因丰度总是比nirK基因丰度高，说明nirS型反硝化细菌更能适应富氧灌溉池塘水质环境。3类反硝化基因丰度与环境参数中的DO浓度和pH值呈显著正相关（P＜0.01）。综上，富氧条件下灌溉池塘水质参数尤其是DO浓度昼夜垂向变化促使nirK，nirS和nosZ 3类反硝化细菌丰度变化具有一定的时间效应和垂直空间分层效应，说明在自然条件下也可能有大量的好氧反硝化细菌存在，并且因为长时间受富氧条件驯化，导致反硝化细菌丰度受 DO 浓度变化影响较大，可能会促使不同深度水层断面发生的氮转化过程及其强度具有差异。进而为原位状态下好氧反硝化脱氮的可能性提供了更有利的佐证。该研究为富氧污水在用于农田灌溉使用时提供背景参数。%In China, there are an increasing number of ponds receiving sewage effluents with high concentrations of nitrogen (N) and dissolved oxygen (DO) for agricultural purposes. Therefore, it is worthwhile to understand the essential N transformation processes as well as the characteristics of N-cycling bacteria driving these processes under the condition of high DO concentrations. In order to evaluate the diel vertical variation of denitrifying bacteria in the water column of the ponds during the process of accumulation and storage of sewage, an experiment was carried out to monitor the changes of denitrifying genes abundance and chemical and physical characteristics at the different depths of sewage water reserved in a pond at Jiangsu Academy of Agricultural Sciences, Nanjing, China. The water samples were collected from a sampling site where had the most stable hydrodynamic condition and least disturbance due to the discharge of sewage water during irrigation season. Water samples were collected from the different water depths of 10-30, 90-110 and 150-170 cm, periodically (5 consecutive time points, 12:00 pm, 0:00 am, 4:00 am, 08:00 am and 12:00 pm at the next day), within 24 hours during 15th -16th August 2013. The collected samples were then analyzed with the abundances of denitrifying genes (nirK,nirK, andnosZ) using real-time polymerase chain reaction (PCR), and water parameters including pH, water temperature, illuminance and the concentrations of DO, NO3--N, NH4+-N and PO43--P . The correlations between the changes ofnirK,nirK, andnosZ genes abundance and water parameters were statistically analyzed. The results showed that the strong and persistent stratification formed along vertical profile of sewage water with regards to all above monitored physical and chemical parameters. The relatively higher pH, water temperature, illuminance, and the concentrations of DO and NO3--N were observed in the surface water layers (10-30 cm), decreasing with the increase of water depths. On the contrast, the higher concentrations of NH4+-N and PO43--P were observed in the bottom water, which means that they were increased with the increase of water depths. Due to high photosynthetic activity of microscopic algae in the pond, the concentrations of DO concentration increased during the daylight and decreased during night. Diurnal variation of pH was positively (P<0.001) correlated with DO concentration in the vertical profile of the water column in the pond, with pH in the ponds also followed the diurnal cycle of sunlight intensity. The abundances ofnirK,nirK, andnosZ genes were strongly affected by the diurnal distribution patterns of pH and DO concentrations along vertical profile of water,with significant higher abundance during the daytime while lower abundance during the night at all the different water depths. Heterogeneous distribution of denitrifying bacteria abundances along the vertical profile of sewage water column were also demonstrated, with the least abundances in the bottom water (150-170 cm) and the highest in the surface water column (10-30 cm). Although thenirK andnirS are functionally equivalent, denitrifying bacteria harboring either nitrite reductase seem to be likely not under the same community assembly rules. The abundance of nirS gene was always greater than that ofnirK gene in all water samples, suggesting that the sewage water in this study was more suitable for the growth ofnirS-type denitrifying bacteria. This speculation was consistent with previously identified habitat preferences ofnirS- andnirK-gene bearing organisms. The abundances ofnirK,nirS, andnosZ genes were significantly correlated with DO concentration and pH (P<0.01). These results demonstrated that the diel vertical variability of the water physical and chemical parameters, especially DO concentrations, in the oxygen-enriched sewage water could affect the diel vertical distribution patterns ofnirK,nirS, andnosZ genes abundances. This may suggest that a large number of aerobic denitrifying bacteria could have extensively developed in sewage water under the condition of high DO concentrations during the process of conservation in the pond. These aerobic denitrifying bacteria can have an important impact on the nitrogen transformation processes in the vertical profile of sewage water column.
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