Profiling of key nitrogen converting organisms in wastewater treatment plants with diffused aeration

Maintaining stable nitrification rates in biological nutrient removal (BNR) systems is difficult due to the slow growth rates of nitrifying bacteria and their sensitivity to environmental and operational conditions. Dissolved oxygen (DO) concentration in the aeration tank significantly affects nitrification and nitrifying bacterial growth. Currently, diffused aeration systems are gaining popularity over conventional surface aeration systems due to their advantages like process stability, better control, and lower cost of operation. However, studies regarding the impact of this aeration type on the selection of functional microbial communities in wastewater treatment plants are still lacking. This study focused on investigating the community structure and activity of key nitrogen converting organisms within two different municipal full-scale wastewater treatment plants (WWTP A and WWTP B) operated with fine bubble diffused aeration. WWTP A was relatively a large plant with a flow rate of 71 ML/day and consisted of three parallel BNR systems (reactor 1, 2, and 3), operated using a similar mode whereas WWTP B was relatively a small plant (0.5 ML/day) with a single BNR system. Composite sludge samples from aeration tanks, as well as influent and effluent water samples, were collected monthly from August 2019 to February 2020 and from June 2020 to August 2020. The nutrient removal performance of the plant was estimated from the influent and effluent chemical analysis. Floc structure analysis and sludge volume index were calculated to assess the settling characteristics. In addition, nitrifying bacterial population dynamics and their activities were assessed using quantitative real-time and reverse transcriptase PCR, respectively in relation to selected plant operational (DO, temperature, substrate concentration) conditions. The average ammonia removal at WWTP A was 95±5.6% which correlated with DO concentration in the aeration tank and the nitrification rate of the plant, whereas the WWTP B recorded 98±02% average ammonia removal efficiency with a more stable DO level in this plant. The sludge volume index (SVI) values were below 150mL/g in both plants, indicating good sludge settling under fine bubble diffused aeration. However, the floc structure varied across the reactors during the study period and ranged from small to medium, open to compact, and irregular with occasional filaments branching mainly in WWTP A. The microbial analysis of sludge samples showed that ammonia oxidising bacteria (AOB) 16S rRNA gene abundance was high in all the three reactors in WWTP A as compared with nitrite oxidising bacteria (NOB). In WWTP B, the average 16S rRNA gene copies for NOB were observed to be higher than AOB. In addition, in WWTP A, a negative correlation was found between the AOB 16S rRNA population and DO concentration in reactor 1 (r = -0.40), while a positive correlation was found in reactor 3 (r = 0.47) with no clear correlation in reactor 2 as well as in WWTP B. In both plants, Nitrobacter spp. was the dominant NOB, while the relative abundance of Nitrospira spp. was generally consistent throughout the study. The nxrB copy number was observed to be higher than that of nxrA (encoding for Nitrobacter spp.). The highest amoA copy number was observed when the temperatures were high (22 ⁰C -26.1 ⁰C), implying that increasing temperatures possibly benefited AOB growth. In terms of functional gene expression, a rapid decrease in expression levels of amoA was observed in both plants while the expression levels of nxrB were observed to increase rapidly as the temperature increased. In contrast, expression levels of the nxrA were relatively more consistent throughout the study period in both plants. At WWTP A, there was a positive correlation between AOB expression (amoA) and DO concentration in all reactors (reactor 1: r = 0.49; reactor 2: r = 0.78 and reactor 3: r = 0.32; p = 0.05). However, no clear correlation was found between NOB expression (nxrA and nxrB) and DO concentration. At WWTP B, a negative correlation was observed between nxrA expression levels and DO concentration (r = - 0.34, p = 0.05). However, DO concentration showed no clear correlation with amoA and nxrB expression levels. The phylogenetic analysis of nxrB populations in both the plants also revealed similarities that are closely related to uncultured Nitrospira spp., nitrite oxidoreductase subunit B, which has been implicated in complete nitrification (COMAMMOX). These observations indicate a need for more research effort using next-generation sequencing to identify and quantify novel nitrifying bacterial including COMAMMOX and ANAMMOX in WWTPs that were previously unachievable using conventional molecular techniques. In conclusion, this study revealed that the fine bubble diffused aeration operated at relatively high DO concentration was able to effectively remove ammonia in both plants resulting in stable and high nitrification rates even at different seasons and loading rates. It also promoted compact flocs with good settleability as well as facilitated optimal and diverse functional nitrifying bacterial community structure and activity.