Theses and dissertations (Applied Sciences)

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Evaluation of the effect of influentammonium : nitrite ratio on anammoxreactor efficiency
(2020) Gasa, Nomalanga Petronella; Bux, Faizal; Pillai, Sheena Kumari Kuttan; Awolusi, Oluyemi Olatunji
The anaerobic ammonium oxidation (anammox) process has been recognized as an energy-efficient and cost-effective alternative to the conventional nitrification-denitrification route. The anammox process offers many advantages over the conventional processes such as less oxygen demand, non-requirement of external carbon source, and low operational cost. However, the major limitation of this process is the extremely slow growth rate of anammox bacteria and the need for stringent metabolic and reactor conditions leading to a long start-up period, which hinders its application in wastewater treatment. This study focused on evaluating the effect of key substrates (ammonium and nitrite) on anammox performance (nitrogen (N) removal) and community structure in anaerobic sequencing batch reactors (ASBR). For this, three 1L reactors containing different ammonium: nitrite ratios namely; Reactor 1 (1 NH4 + - N: 1.32 NO2 - -N), Reactor 2 (2 NH4 + -N: 1 NO2 - -N) and Reactor 3 (1 NH4 + -N: 2 NO2 - -N) were operated for 320 days using enriched anammox bacterial seed inoculum. The N removal performance of the reactors was assessed over time based on chemical and microbial analysis. From the results, the highest nitrogen removal efficiency (NRE) was observed in Reactor 3 containing high NO2 - -N (68.1 ± 7.7 %), followed by Reactor 1 containing the reported anammox stoichiometric substrate ratio (66.3 ± 13.3 %) and Reactor 2 containing high NH4 + -N (64.1 ± 7.2 %) on the 320th day of reactor operation. By using different substrate ratios, a significant variation (α= 0.05; P= 0.0004) in NRE in the three reactors was observed. Overall, the observed NO2 - - N (consumed)/NH4 + -N (removed), NO3 - -N (produced)/NH4 + -N (removed) ratios in Reactor 3 (1.38 ± 0.35 and 0.51 ± 0.34) was closer to the reported anammox stoichiometry ratio compared to Reactor 1 (0.88 ± 0.35 and 0.91 ± 0.48) and Reactor 2 (0.69 ± 0.32 and 0.72 ± 0.26) indicating a better anammox enrichment in Reactor 3. The inhibitory impact of free ammonia (FA) and free nitrous acid (FNA) concentration was monitored throughout the operational period. The FA concentration did not have a negative effect on anammox bacteria and AOB since the observed FA inhibitory concentration was below the reported inhibitory concentration of 1700 µg/L for anammox bacteria in all three reactors. As for FNA, Reactor 3 recorded the highest FNA concentrations (27.3 – 27.4 µg HNO2 - -N/L) throughout the study period. This FNA concentration did not negatively affect anammox bacteria on the 170th day, since anammox population was increased. However, long-term exposure resulted in anammox inhibition on the 320th day indicated by reduction of anammox bacteria. Whereas, nitrite oxidising bacteria (NOB) were not negatively affected by the observed FNA concentration, since their activity and growth was observed throughout the operation. As for Reactor 1 and 2, the FNA concentration (5.5 – 5.9 µg HNO2 - -N/L) was below inhibitory concentration on the 170th day. However, on the 320th day, the FNA concentration (6.2 – 7.3 µg HNO2 - -N/L) was above the reported inhibitory value resulting in anammox inhibition. A detailed exploration of the changes in the microbial community structures within the three reactors were studied by quantitative polymerase chain reaction (qPCR), sequencing and phylogenetic analyses. Using qPCR, Reactor 3 (1:2) with high NH4 + -N concentration showed high abundance of anammox bacteria followed by Reactor 2 (2:1) with high NO2 - -N concentration and Reactor 1 (1:1.32) having balanced NH4 + -N: NO2 - -N respectively on the 170th day. Thereafter, a shift from anammox bacteria abundance towards proliferation of AOB and NOB was observed on the 320th day. The AOB population was favoured by the fluctuating DO concentrations (0.39 ± 0.19 – 0.49 ± 0.20 mg/L). High AOB population observed in Reactor 1 (1:1.32) followed by Reactor 3 (1:2) and Reactor 2 (2:1) on 170th and 320th day. The NOB population was high in Reactor 3 (1:2) followed by Reactor 1 (1:1.32) and Reactor 2 (2:1) respectively throughout the operational period. High throughput sequencing analysis further showed a shift in the microbial community structure on 170th day with an increase in phylum Planctomycetes population from 0.76 % to 3.30 % in Reactor 1, 21. 32 % in Reactor 2 and 22.26 % in Reactor 3. The population of Proteobacteria increased from 6.38 % to 6.70 % in Reactor 1, 21.63 % in Reactor 2 and 21.73 % in Reactor 3. On the 320th day, Planctomycetes population decreased drastically to 2.84 %, 0.36 % and 4.91 % in Reactors 1, 2 and 3, respectively. Whereas Proteobacteria population further increased to 28.95 %, 24.15 % and 23.86 % in Reactors 1, 2 and 3, respectively. The Nitrospira population were below 0.10 % on the 170th day, however, an increase was observed on the 320th day from 0.01 % to 2.84 %, 7.38 % and 1.09 % in Reactors 1, 2 and 3, respectively which are in accordance with the qPCR results. In conclusion, different substrate ratios showed a significant influence on the overall N removal performance as well as on the selection of nitrifiers during the initial 170 days of operation. However, the long term operation of the reactors negatively affected the performance as well as community structure irrespective of the ratio used. Furthermore, the intermittent spike in DO and FNA concentrations (above inhibitory levels) could have affected the growth of anammox bacteria adversely. A further study based on continuous reactor operation is recommended for further verification of the results and prediction of unstable reactor episodes and possible process inhibitions in real-time.
Profiling of key nitrogen converting organisms in wastewater treatment plants with diffused aeration
(2022-09) Kumalo, Puseletso Constance; Bux, Faizal; Pillai, Sheena Kumari Kuttan; Awolusi, Oluyemi Olatunji
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.