Wastewater treatment plants (WWTPs) are required to remove ammonium (NH4+) from wastewater due to its oxygen demand and toxicity to the aquatic organisms. Ammonium is removed in the activated sludge treatment system by nitrification and denitrification processes. Nitrification is the oxidation of NH4+ to nitrate (NO3-) by autotrophic nitrifying bacteria which use carbon dioxide (CO2) as a carbon source for growth. These bacteria grow slowly with low nitrification rates limiting WWTPs capacity. In this research it was hypothesized that supplying higher concentrations of CO2 during aeration increases nitrification rates, resulting in a reduction of the solids retention time (SRT). This hypothesis was tested with two lab-scale sequencing batch reactors seeded with sludge from a full-scale activated sludge WWTP and fed synthetic wastewater. The control reactor was aerated with regular air (0.03% CO2) and the experimental reactor was aerated with air containing 1% CO2. Ammonium and NO3- were measured online to determine the nitrification rates. Samples for solids and chemical oxygen demand (COD) determination were collected to evaluate the system performance. Supplying CO2 to the experimental reactor throughout the entire react cycle resulted in proliferation of filamentous bacteria, poor settling, and washout of the biomass. However, nitrate formation rates in the experimental reactor were 3 times higher than the control before washout occurred. In a subsequent experiment, CO2 was supplied to the experimental reactor only during the last 5 hours of the cycle, resulting in excellent settling and nitrification rates 6 times higher than in the control. A confirmatory experiment was conducted that lowered the SRT from 8 days to 6, 4, and 2 days. Nitrate formation rates were up to 12 times higher in the experimental reactor compared to the control, with an average of 4 times higher. Additionally, the sludge volume index (SVI) suggested a positive impact of CO2 on settling performance. No impact of CO2 on COD removal was observed. The results obtained suggest a positive effect of CO2 on the nitrate formation and settling performance in the activated sludge system, indicating that nitrification can be achieved at low SRTs which might optimize WWTPs capacity.

Submitted in complete fulfillment for the degree of Doctor of Philosophy (Biotechnology), Durban University of Technology, Durban, South Africa, 2016. / Seasonal nitrification breakdown is a major problem in wastewater treatment plants which makes it difficult for the plant operators to meet discharge limits. The present study focused on understanding the seasonal impact of environmental and operational parameters on nitrifiers and nitrification, in a biological nutrient removal wastewater treatment works situated in the midlands of KwaZulu Natal. Composite sludge samples (from the aeration tank), influent and effluent water samples were collected twice a month for 237 days. A combination of fluorescent in-situ hybridization, polymerase chain reaction (PCR)-clone library, quantitative polymerase chain reaction (qPCR) were employed for characterizing and quantifying the dominant nitrifiers in the plant. In order to have more insight into the activated sludge community structure, pyrosequencing was used in profiling the amoA locus of ammonia oxidizing bacteria (AOB) community whilst Illumina sequencing was used in characterising the plant’s total bacterial community. The nonlinear effect of operating parameters and environmental conditions on nitrification was also investigated using an adaptive neuro-fuzzy inference system (ANFIS), Pearson’s correlation coefficient and quadratic models. The plant operated with higher MLSS of 6157±783 mg/L during the first phase (winter) whilst it was 4728±1282 mg/L in summer. The temperature recorded in the aeration tanks ranged from 14.2oC to 25.1oC during the period. The average ammonia removal during winter was 60.0±18% whereas it was 83±13% during summer and this was found to correlate with temperature (r = 0.7671; P = 0.0008). A significant correlation was also found between the AOB (amoA gene) copy numbers and temperature in the reactors (α= 0.05; P=0.05), with the lowest AOB abundance recorded during winter. Sanger sequencing analysis indicated that the dominant nitrifiers were Nitrosomonas spp. Nitrobacter spp. and Nitrospira spp. Pyrosequencing revealed significant differences in the AOB population which was 6 times higher during summer compared to winter. The AOB sequences related to uncultured bacterium and uncultured AOB also showed an increase of 133% and 360% respectively when the season changed from winter to summer. This study suggests that vast population of novel, ecologically significant AOB species, which remain unexploited, still inhabit the complex activated sludge communities. Based on ANFIS model, AOB increased during summer season, when temperature was 1.4-fold higher than winter (r 0.517, p 0.048), and HRT decreased by 31% as a result of rainfall (r - 0.741, p 0.002). Food: microorganism ratio (F/M) and HRT formed the optimal combination of two inputs affecting the plant’s specific nitrification (qN), and their quadratic equation showed r2-value of 0.50. This study has significantly contributed towards understanding the complex relationship between the microbial population dynamics, wastewater composition and nitrification performance in a full-scale treatment plant situated in the subtropical region. This is the first study applying ANFIS technique to describe the nitrification performance at a full-scale WWTP, subjected to dynamic operational parameters. The study also demonstrated the successful application of ANFIS for determining and ranking the impact of various operating parameters on plant’s nitrification performance, which could not be achieved by the conventional spearman correlation due to the non-linearity of the interactions during wastewater treatment. Moreover, this study also represents the first-time amoA gene targeted pyrosequencing of AOB in a full-scale activated sludge is being done. / D

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