In recent years considerable interest has been shown in the diversity of ammonia-oxidizing bacteria in soil communities. The majority of the research has been carried out in Northern Europe where soils have received high atmospheric inputs of nitrogen over the past two centuries. In contrast, although much work has been conducted on nitrogen cycling processes in nitrogen limited forest ecosystems in western North America, no studies have examined the characteristics of ammonia-oxidizing communities in those environments. I was interested in measuring nitrification potential along a high-elevation temperate meadow-to-forest gradient, and characterizing the ammonia-oxidizing communities along that gradient using both molecular and culturing methods. Two experimental sites (Lookout and Carpenter) were chosen in the H.J. Andrews Experimental Forest, located in the western Cascade Range of Oregon, at elevations of approximately 1500 meters. Although nitrification potential rates (NPRs) between sites were not significantly different (P=0.544), variation was observed both within and between sites for specific vegetation types. NPRs were significantly lower in forest (F) soil samples than in meadow (M) soil samples, averaging 5 and 2% of meadow NPRs at Lookout and Carpenter, respectively. In meadow soil samples, most probable number (MPN) population densities of ammonia-oxidizers ranged from 0.6 to 2.6 x 10⁴ cells gram⁻¹ of oven dry soil and 0.9 x 10³ to 1.1 x 10⁵ cells g⁻¹ OD soil at Lookout and Carpenter, respectively. In forest soil samples, population densities ranged from undetectable to 1.1 x 10⁴ cells g⁻¹ OD soil, and 0.9 x 10² to 2.3 x 10³ cells g⁻¹ OD soil at Lookout and Carpenter, respectively. Microbial community DNA was amplified using primers to the ammonia monooxygenase subunit A. Terminal restriction fragments polymorphism analysis with three different restriction enzymes (CfoI, TaqI, and AluI) revealed community profiles dominated by Nitrosospira species. One fragment from CfoI (66 bp) and one fragment from AluI (392-bp) were prominent in 47 soil samples from both sites, and represented between 32 to 100% of the Genescan fragment analyses of PCR products. A full length fragment from AluI digests (491-bp), and three fragments from CfoI (68, 100, and 135- bp) were found sporadically in fewer soil sample T-RFLPs, and within those samples represented smaller percentages of total peak areas. The CfoI 135-bp fragment length was associated primarily with M and meadow/forest (M/F) soils where it was observed in approximately 58 and 100% of the respective transect locations. Eight isolates recovered from soil samples were analyzed using the same molecular methods as the field samples. The T-RFLP patterns of the isolates corresponded with many of those found in the community fingerprints. Four unique amoA sequences were identified among these isolates, including one that possessed the dominant T-RFLP amoA fingerprint in soil samples. This sequence shared 99.8% similarity with Nitrosospira sp. Ka4, a cluster 4 ammonia oxidizer isolated in Norway. Sequence analysis phylogenetically associated the other three isolates (with unique amoA sequences) near Nitrosospira sp. Nsp 1 and Nitrosospira briensis, both cluster 3 ammonia oxidizers. Cloning and sequencing of soil DNA confirmed that ammonia oxidizers with these amoA sequences were present in the soil samples. Two additional amoA sequences were identified in clones that were 95% similar and paraphylogenetically positioned between representatives of clusters 3 and 4. So far, these sequences have not been found in any of the isolates analyzed. / Graduation date: 2003

<p> This report examines the dynamic response of a 0. 5 metre pilot scale rotating biological contactor when operated under transient influent conditions. Experimental data are presented for two modes of operation: carbon oxidation and carbon oxidation plus nitrification. During the carbon oxidation experimental runs, a 2.0 metre RBC was operated in parallel with the 0.5 metre RBC, thus allowing comparisons in the performance of the units. </p> <p> When the 0. 5 metre RBC was operated under nitrifying conditions, it was found that the effluent filterable TKN responded positively to influent variations in filterable TKN loading, TKN concentration and hydraulic loading. Transfer f unction noise models were developed which successfully predicted the time varying effluent TKN response. The response in effluent filterable TKN was predicted most precisely by influent TKN loading. Models based on influent TKN concentration and flow were not as precise in predicting effluent response. The effluent response of the 0.5 metre RBC was found to be greater than the response of activated sludge pilot units when operated at similar levels of removal. </p> <p> When the 0. 5 metre RBC was operated in the carbon oxidation 'mode, significant effluent responses were observed for carbon loading and concentration. Little correlation was found between influent flow and effluent carbon concentration. Operating under identical conditions, the 2.0 m RBC showed significant responses to carbon loading, carbon concentration and flow. </p> / Thesis / Master of Engineering (MEngr)

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

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es. / Graduation date: 2012

La bio-augmentation des stations d’épuration est parfois envisagée pour faire face à des situations transitoires au cours desquelles la capacité nitrifiante des boues activées est insuffisante pour traiter efficacement la charge reçue par l’installation. Le succès de cettestratégie repose sur l’activité nitrifiante de la biomasse ajoutée ainsi que sur sa capacitéd’adaptation et de maintien dans les boues activées en place.L’objectif de ce travail était d’évaluer la possibilité d’adaptation des micro-organismes d’une solution commerciale nitrifiante dans une station d’épuration municipale à très faible charge.Les populations nitrifiantes composant la solution étudiée ont été comparées à celles des boues activées de treize stations d’épurations, échantillonnées en été et en hiver. Une démarche couplant des outils de la biologie moléculaire et du génie des procédés a été mise en oeuvre afin d’analyser et de comparer ces populations selon trois axes : diversité, abondance et activité. Cette comparaison a été complétée par une expérience de bio-augmentation en réacteur fermé. D’après les profils obtenus en DGGE, les communautés nitrosantes des stations d’épuration à très faible charge sont généralement dominées par une même espèce appartenant au groupe Nitrosomonas oligotropha.Les biomasses nitrosantes et nitratantes ont été chacune estimées entre 0,5 et 3,5 % de la biomasse totale des boues activées par marquage FISH. La solution commerciale comporteune seule espèce nitrosante, du groupe N. europaea, qui représente 45 % de sa biomasse.Les analyses cinétiques, réalisées par suivi direct des vitesses de réaction (analyses chimiques) et/ou par respirométrie, indiquent que N. europaea adopte une stratégie de type –r contrairement aux bactéries autochtones des boues activées (stratégie de type –K). Ces résultats expérimentaux laissent supposer une faible capacité d’adaptation de la biomasse nitrifiante de cette solution commerciale aux conditions environnementales des stationsd’épuration à très faible charge. En revanche, l’expérience de bio-augmentation a montré sa capacité d’intégration aux flocs, et le maintien de son activité lors de son mélange à des boues activées. / Bio-augmentation is sometimes proposed to cope with transient situations in wastewatertreatment plants, when the nitrifying capacity of the autochthonous biomass is insufficient totreat the nitrogen load.The success of such a strategy is conditioned by the nitrifying activity of the added biomass,its adaptation to local conditions and its residence time in the system.This work aimed at evaluating the adaptation of nitrifying microorganisms from a commercialsolution in very low loaded municipal wastewater treatment plant. Nitrifying populations ofthe studied solution were compared to those of thirteen wastewater treatment plants (activatedsludge), observed in summer and in winter. The analysis and the comparison of the biomasseswere performed using tools from two disciplines: molecular biology and processengineering. Three points were especially characterised: the diversity, abundance and activityof the biomasses. A bio-augmentation experiment performed in a batch reactor completed thecomparison. DGGE profiles showed that Ammonia Oxidising Bacteria (AOB) communities from lowloaded wastewater treatment plants are mostly dominated by the same species related to theNitrosomonas oligotropha.The abundance of ammonia oxidising bacteria and of nitrite oxidising bacteria, estimatedusing the FISH technique, was almost similar, ranging from 0.5 to 3.5 % of the total biomass.The AOB species present in the commercial solution is identified as Nitrosomonas europaea,and represents 45 % of the solution’s total biomass.Kinetic analyses carried out by chemical analysis and/or by respirometry indicate that N.europaea behave as –r strategist, whereas activated sludge’s bacteria are –K strategist. These results suggest a slight adaptation capacity of the commercial solution’s nitrifyingbiomass to environmental conditions prevailing in low loaded municipal wastewater treatmentplants. On the other hand, bio-augmentation experiment showed the capacity of the addedmicroorganisms to integrate the activated sludge flocs and to maintain its activity when addedto activated sludge.