Faculty of Applied Sciences
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Item Evaluation of the effect of influentammonium : nitrite ratio on anammoxreactor efficiency(2020) Gasa, Nomalanga Petronella; Bux, Faizal; Pillai, Sheena Kumari Kuttan; Awolusi, Oluyemi OlatunjiThe 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.Item Determining the efficiency of the anammox process for the treatment of high- ammonia influent wastewater(2017-08) Gokal, Jashan; Bux, Faizal; Stenström, Thor-Axel; Kumari, Sheena K.Domestic wastewater contains a high nutrient load, primarily in the form of Carbon (C), Nitrogen (N), and Phosphorous (P) compounds. If left untreated, these nutrients can cause eutrophication in receiving environments. Biological wastewater treatment utilizes a suspension of microorganisms that metabolize this excess nutrient load. Nitrogen removal in these systems are due to the synergistic processes of nitrification and denitrification, each of which requires its own set of operating parameters and controlling microbial groups. An alternative N-removal pathway termed the anammox process allows for total N-removal in a single step under anoxic conditions. This process, mediated by the anammox bacterial group, requires no organic carbon, produces negligible greenhouse gases and requires almost 50 % less energy than the conventional process, making it a promising new technology for efficient and cost-effective N-removal. In this study, a sequencing batch reactor (SBR) was established for the autotrophic removal of N-rich wastewater through an anammox-centric bacterial consortia. The key microbial members of this consortia were characterized and quantified over time using molecular methods and next generation sequencing to determine if the operational conditions had any effect on the seed inoculum population composition. Additionally, local South African wastewater treatment plants were screened for the presence of anammox bacteria through 16S rRNA amplification and enrichment in different reactor types. A 3 L bench scale SBR was inoculated with active biomass (~ 5 % (v/v)) sourced from a parent anammox enrichment reactor, and maintained at a temperature of 35 °C ± 1 °C. The reactor was fed with a synthetic wastewater medium containing no organic C, minimal dissolved oxygen (< 0.5 mg/L), and N in the form of ammonium and nitrite in the ratio of 1:1.3. The reactor was operated for a period of 366 days and the effluent ammonium, nitrite and nitrate were measured during this period. The hydraulic retention time was controlled at 4.55 days from Day 1 to Day 250, and thereafter shortened to 1.52 days from Day 251 to Day 360 due to an increased nitrogen removal rate (NRR). During Phase I of operation (Day 1 to Day 150), the reactor performance gradually increased up to an NRR of ~160 mg N/day. During Phase II (Day 151 to Day 250), the overall reactor performance decreased with the NRR decreasing to ~90 mg N/day, while Phase III (Day 251 to Day 366) displayed a gradual recovery of NRR back to the reactor optimum of ~160 mg N/day. The accumulation of nitrate in the effluent during the latter parts of Phase II and Phase III, coupled with oxygen ingress (~2.1 mg/L) in the same period, indicated that it was not the anammox pathway that was dominating N-removal within the reactor, but more likely the second half of the nitrification pathway mediated by the nitrite oxidizing bacteria (NOB). This was further confirmed through molecular analysis, which indicated that the bacterial population had shifted significantly over the course of reactor operation. Quantitative PCR methods displayed a decrease in all the key N-removing population groups from Day 1 to Day 140, and a marginal increase in anammox and aerobic ammonia oxidizing bacteria from Day 140 – Day 260. From Day 300 onwards, NOB had started dominating the system, simultaneously suppressing the growth of other N-removing bacterial groups. Despite this, the NRR peaked during this period, indicating an alternative mechanism for ammonia removal within the reactor system. A total population analysis using NGS was also performed, which corroborated the QPCR results and displayed a population shift away from anammox bacteria towards predominantly NOB and members of the phylum Chloroflexi. The proliferation of aerobic NOB and Chloroflexi, and the suppression of anammox bacteria, indicated that DO ingress was indeed the primary cause of the population shift within the reactor. Despite this population shift, N-removal within the reactor remained high. New pathways have recently emerged which implicate these two groups as potential N oxidizers, with specific NOB groups showing the ability for oxidation of ammonia through the comammox process, and members of the Phylum Chloroflexi being capable of nitrite reduction. This could imply that an alternate pathway was responsible for the majority of N-removal within the system, in addition to the anammox and conventional nitrification pathways. Additionally, in an attempt to detect a local anammox reservoir, eleven wastewater systems from around South Africa were screened for the presence of anammox bacteria. Through direct and nested PCR-based screening, anammox bacteria was not detectable in any of the activated sludge samples tested. Based on the operating conditions of the source wastewater systems, a subset of three sludge samples were selected for further enrichment. After 60-110 days of enrichment in multiple reactor configurations, only one reactor sample tested positive for the presence of anammox bacteria. Although this result indicates that anammox bacteria might not be ubiquitous within every biological wastewater system, it is more likely that anammox bacteria might only be present at undetectable levels, and that an extended enrichment prior to screening is necessary for a true representation of anammox bacterial prevalence in an environmental sample.