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Depuis la découverte d’archées capables d’oxyder l’ammoniac en milieu aérobie, de nombreuses études ont mesuré en simultané les taux de nitrification et la diversité des organismes oxydant l’ammoniac dans la colonne d’eau des milieux marins. Malgré l’importance globale des lacs d’eau douce, beaucoup moins d’études ont fait la même chose dans ces milieux. Dans cette étude, nous avons évalué l’importance de la nitrification et caractérisé la communauté microbienne responsable de la première étape limitante de la nitrification dans un lac tempéré durant une année entière. L’utilisation de traceur isotopique 15NH4 nous a permis de mesurer des taux d’oxydation d’ammoniac à deux profondeurs dans la zone photique tout au long de l’année. Les taux d’oxydation d’ammoniac varient de non détectable à 333 nmol L-1 j-1 avec un pic d’activité sous la glace. De toutes les variables environnementales mesurées, la concentration d’ammonium dans la colonne d’eau semble avoir le plus grand contrôle sur les taux d’oxydation d’ammoniac. Nous avons détecté la présence d’archées (AOA) et de bactéries oxydante d’ammoniac (BOA) à l’aide de tests par réaction en chaîne de la polymérase (PCR) ciblant une partie du gène ammoniac monoxygénase (amoA). Les AOA et les BOA ont été détectées dans la zone photique du lac, cependant seules les AOA étaient omniprésentes durant l’année. Le séquençage du gène amoA des archées révèle que la majorité des AOA dans le lac sont membres du groupe phylogénétique Nitrosotalea (également appelé SAGMGC-1 ou groupe I.1a associé), ce qui confirme la pertinence écologique de ce groupe dans les eaux douces oligotrophes. Globalement, nos résultats indiquent l’hiver comme étant un moment propice pour l’oxydation de l’ammoniac dans les lacs tempérés. Cette étude fournit un point de référence pour la compréhension du processus d’oxydation de l’ammoniac dans les petits lacs oligotrophes. / Since the discovery that some archaea are able to oxidize ammonia aerobically, several studies have focused on measuring nitrification rates and identifying the diversity of planktonic ammonia oxidizers in marine systems. Despite the global importance of freshwater lakes, far fewer studies have done the same in these ecosystems. Here we investigated the importance of nitrification and characterize the microbial community catalyzing the first rate-limiting step of nitrification over an annual cycle in a temperate lake. The measurements of ammonia oxidation rates, using the 15NH4+ isotope tracer method, at two depths in the photic zone show that this process occurred throughout the entire year in the lake. Rates of ammonia oxidation ranged from undetectable to 333 nmol L-1 d-1 with a peak of activity during winter. Off all environmental variables measured, ammonium concentrations in the water-column seem to have the strongest effect on the magnitude of ammonia oxidation rates. We detected the presence of ammonia-oxidizing archaea (AOA) and bacteria (AOB) using polymerase chain reaction (PCR) assays targeting part of the ammonia monooxygenase (amoA) gene. Both AOA and AOB were detected in the photic zone of the lake, although only AOA were omnipresent over the year. The sequencing of archaeal amoA genes reveals that most of the AOA in the lake are members of the Nitrosotalea cluster (also referred as SAGMGC-1 or group I.1a associated), which confirms the ecological relevance of this cluster in oligotrophic freshwaters. Altogether, our results indicate that winter may be a critical time for ammonia oxidation in temperate lakes and provide a baseline for the understanding of ammonia oxidation in small oligotrophic lakes.

Nitrogen cycling processes can be tracked using quantitative Polymerase Chain Reaction (qPCR) to determine the presence and qReverse Transcriptase-PCR (qRT-PCR) to determine expression of key genes, or ‘biological markers’, for nitrogen metabolism. Nitrification is catalyzed in part, by two enzymes: ammonia monooxygenase (AMO; NH3 NH2OH) and nitrite oxidoreductase (NXR; NO2- NO3-). For denitrification, four enzymes act sequentially: nitrate reductase (NAR/NAP; NO3- NO2-), nitrite reductase (NIR; NO2- NO), nitric oxide reductase (NOR; NO  N2O), and nitrous oxide reductase (NOS; N2O  N2). A principle of wastewater treatment (WWT) is to remove excess nitrogen by taking advantage of natural nitrogen cycling or biological nitrogen removal (BNR). This process involves using microorganisms to bring influent ammonia through nitrification and denitrification to release nitrogen gas, which does not contribute to eutrophication. A novel shortcut nitrogen removal configuration could increase nitrogen removal efficiency by promoting nitritation/denitritation, reducing the classic nitrogen cycle by removing the redundant oxidation/reduction step to nitrate (NO3-). Here, three nitrogen transformations were used to track the three main phases in the nitrogen cycle; ammonia monooxygenase for nitrification, nitrite oxidoreductase for shortcut, and nitrous oxide reductase for denitrification. Primers for qPCR and qRT-PCR were designed to capture as much sequence diversity as possible for each step. Genes from bacteria known to perform the nitrogen transformations of interest (amoA, nxrB, nosZ) were used to BLAST-query the Integrated Microbial Genomes & Microbiomes database (img.jgi.doe.gov) to find homologs from organisms commonly found in WWT. These sequences were then aligned to find regions sufficiently conserved for primer design. These PCR primers were tested against standards for each gene and used to track nitrogen transformation potential and expression in a novel lab-scale algal photo-sequencing batch reactor which promotes shortcut nitrogen removal from wastewater across three solids retention times (SRT, or mean cell residence time); 5, 10 and 15 days. SRT 15 had the greatest total nitrogen removal with nitritation and denitritation observed. Nitrate was not detected in the first cycle and shortcut nitrogen removal was supported by low levels of nxrB genes and transcripts. Simultaneous nitrification/denitrification was supported by elevated concentrations of nosZ during the light period and less nitrite produced than ammonium consumed. Nitritation was predominantly performed by Betaproteobacteria amoA and nitrous oxide reduction was predominantly from nosZ group I (Proteobacteria-type).

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