Faculty of Applied Sciences
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Item Impact of chemical oxygen demand to nitrogen ratio on anammox bacterial growth in an up-flow anaerobic sludge blanket reactor(2023) Msimango, Sandile Simiso; Kumari, Sheena; Nasr, Mahmoud; Bux, FaizalThe anaerobic oxidation of ammonium (ANAMMOX) process has been suggested as an economical and innovative means of removing nitrogen from wastewater. Nevertheless, very few studies have evaluated the effect of the chemical oxygen demand (COD) to nitrogen (N) ratio on bacterial communities in an ANAMMOX-mediated system. Heterotrophic bacteria can readily outcompete the slow-growing ANAMMOX bacteria in the presence of organic carbon. This study examined the effect of the organic carbon to nitrogen (C/N) ratio on the performance of ANAMMOX in an upflow sludge blanket reactor using synthetic wastewater as the feedstock. Two UASB reactors (UASB-A and UASB-B) were seeded with biomass from a labscale ANAMMOX reactor and operated for a period of 593 days. Both reactors were operated using similar operational conditions during the enrichment phase (0-400 days). Thereafter, the addition of organic carbon in the medium altered the C/N ratio of one of the reactors (UASBB). During this period, UASB-A served as a control reactor. A CN ratio of 1.0, 1.5, and 2.0 was achieved in the UASB B reactor by increasing the organic carbon concentration every 60 days. The reactors were analyzed at three-day intervals per week for nitrogen and COD removal efficiency. The quantitative PCR method was used to detect the dominant N-removing organisms within both reactors at different phases. In addition, cDNA quantification or reverse transcriptase qPCR (RT-qPCR) was also conducted to determine the dominant and active nitrifying communities. The results indicated that when the C/N ratio is 1.0, almost complete removal of NH4 + -N is observed (92%), and nitrogen removal efficiency (NRE) is approximately 82%. The ratios of ΔNO2 - /ΔNH4 + and ΔNO3 - /ΔNH4 + ratios during this phase (C/N=1) fluctuated from >1.25 to <1.6 and from >0.35 to <0.45 <0.11 to >1.6, respectively, which was within the range of the expected ANAMMOX stoichiometric ratio. In addition, when the C/N ratio was increased from 1 to 1.5, NRE rose from 82 to 88%. However, a decrease of NRE to 83% was observed when the C/N ratio was further increased to 2. The quantitative PCR results showed an increase in total bacteria from 1.4 × 106 copies/µL to 2.3× 106 copies/µL, and 2.4× 106 copies/µL as the ratio of C/N increased from 1.0 to 1.5 and thereafter to 2, respectively. ANAMMOX bacteria showed an increase from 16 × 103 copies/µL to 6.5× 10 4 copies/µL, and 2.06 × 105 copies/µL when the C/N ratio was increased from 1 to 1.5, and 2, respectively. The cDNA analysis further showed an increase of ANAMMOX bacteria transcript abundance from 4.6 × 104 copies/µL to 2.52× 106 copies/µL with an increase in C/N ratio to 1.5. Subsequently, a decrease in ANAMMOX bacteria transcript abundance to 1.09 × 106 copies/µL was observed when the C/N ratio was further increased to 2. The expression of the hzo gene encoding for hydrazine dehydrogenase (HDH), which catalyses the oxidization of the unique ANAMMOX intermediate hydrazine to N2 was 169 folds of expression, which was very high at C/N=1, but showed a decrease to 39 folds expression at C/N=1.5. Almost complete inhibition of hzo gene was observed when the C/N ratio was further increased to 2. Based on chemical analysis, it was further confirmed that the decrease of both ANAMMOX and AOB abundance at a higher C/N ratio caused an increase in effluent NH4 + -N concentrations. In conclusion, the study has shown that a higher C/N ratio could significantly affect the overall nitrogen removal rate and the activity of the diverse microbial populations, more specifically the ANAMMOX bacterial activity.Item 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 OlatunjiMaintaining 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.