Protecting the quality and quantity of our water resources requires advanced treatment technologies capable of removing nutrients from wastewater. This research work investigated the capability of one such technology, a hollow-fiber membrane-aerated biofilm reactor (HFMBR), to achieve completely autotrophic nitrogen removal from a wastewater with high nitrogen content. Because the extent of oxygenation is a key parameter for controlling the metabolic processes that occur in a wastewater treatment system, the first part of the research investigated oxygen transfer characteristics of the HFMBR in clean water conditions and with actively growing biofilm. A mechanistic model for oxygen concentration and flux as a function of length along the non-porous membrane fibers that comprise the HFMBR was developed based on material properties and physical dimensions. This model reflects the diffusion mechanism of non-porous membranes; namely that oxygen follows a sorption-dissolution-diffusion mechanism. This is in contrast to microporous membranes in which oxygen is in the gas phase in the fiber pores up to the membrane surface, resulting in higher biofilm pore liquid dissolved oxygen concentrations. Compared to offgas oxygen analysis from the HFMBR while in operation with biofilm growing, the model overpredicted mass transfer by a factor of approximately 1.3. This was in contrast to empirical mass transfer coefficient-based methods, which were determined using either bulk aqueous phase dissolved oxygen (DO) concentration or the DO concentration at the membrane-liquid interface, measured with oxygen microsensors. The mass transfer coefficient determined with the DO measured at the interface was the best predictor of actual oxygen transfer under biofilm conditions, while the bulk liquid coefficient underpredicted by a factor of 3. The mechanistic model exhibited sensitivity to parameters such as the initial lumen oxygen concentration (at the entry to the fiber) and the diffusion coefficient and partitioning coefficients of oxygen in the silicone membrane material. The mechanistic model has several advantages over empirical-based methods. Namely, it does not require experimental determination of KL, it is relatively simple to solve without the use of advanced mathematical software, and it is based upon selection of the membrane-biofilm interfacial DO concentration. The last of these is of particular importance when designing and operating HFMBR systems with redox (aerobic/anoxic/anaerobic) stratification, because the DO concentration will determine the nature of the microenvironments, the microorganisms present, and the metabolisms that occur. During the second phase of the research, the coupling of two autotrophic metabolisms, partial nitrification to nitrite (nitritation) and anaerobic ammonium oxidation, was demonstrated in a single HFMBR. The system successfully treated a high-strength nitrogen wastewater intended to mimic a urine stream from such sources as extended space missions. For the last 250 days of operation, operating with an average oxygen to ammonia flux (J_O₂/J_NH₄⁺) of 3.0 resulted in an average nitrogen removal of 74%, with no external organic carbon added. Control of nitrite-oxidizing bacteria (NOB) presented a challenge that was addressed by maintaining the J_O₂/J_NH₄⁺ below the stoichiometric threshold for complete nitrification to nitrate (4.57 g O₂ / g NH₄⁺). The DO-limiting condition resulted in formation of harmful gaseous emissions of nitrogen oxides (NO, N2O), which could not be prevented by short-term control strategies. Controlling JO2/JNH4+ prevented NOB proliferation long enough to allow an anaerobic ammoniaoxidizing bacteria (AnaerAOB) population to develop and be retained for >250 days. Addition of a supplemental nutrient solution may have contributed to the growth of AnaerAOB by overcoming a possible micronutrient deficiency. Disappearance of the gaseous nitrogen oxide emissions coincided with the onset of anaerobic ammonium oxidation, demonstrating a benefit of coupling these two autotrophic metabolisms in one reactor. Obvious differences in biofilm density were evident across the biofilm depth, with a region of low density in the middle of the biofilm, suggesting that low cell density or exocellular polymeric substances were primarily present in this region, Microbial community analysis using fluorescence in situ hybridization (FISH) did not reveal consistent trends with respect to length along the fibers, but radial stratification of aerobic ammonia-oxidizing bacteria (AerAOB), NOB, and AnaerAOB were visible in biofilm section samples. AerAOB were largely found in the first 25% of the biofilm near the membrane, AnaerAOB were found in the outer 30%, and NOB were found most often in the mid-depth region of the biofilm. This community structure demonstrates the importance of oxygen availability as a determinant of how microbial groups spatially distribute within an HFMBR biofilm. The combination of these two aspects of the research, predictive oxygen transfer capability and the effect of oxygen control on performance and populations, provides a foundation for future application of HFMBR technology to a broad range of wastewaters and treatment scenarios. / Ph. D.

Symbiosis is commonly present in marine invertebrates. Many corals and sponges have symbiotic algae or bacteria. In the previous studies of the sponge Cinachyra australiensis, 85% of the bacteria associated with the sponge have high similarity (88.65%) with the symbiotic chemoautotrophic sulfur-oxidizing bacteria of the deep-sea hydrothermal vent mussel, Solemya reidi. This study aims to investigate the localization of the chemoautotrophic sulfur-oxidizing bacteria associated with Cinachyra australiensis. The Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) large-subunit genes for autotrophic organisms were amplified by polymerase chain reaction from the sponge samples. The phylogenetic relationship of the RubisCO large subunit genes was analyzed. A total of 26 clones were selected and sequenced. They could be divided into two groups. One (9 clones) belongs to form I type IB (cynobacteria and green algae). The other (17 clones) belongs to form II type IA (chemoautotrophic symbiotic bacteria). The location of the sulfur-oxidizing chemoautotrophic bacteria was shown to be intracellular symbiosis within the mesoglial cells by fluorescence in situ hybridization.

Boreal peatlands are important ecosystems to the global carbon cycle. Although they cover only 3% of the earth's land surface area, boreal peatlands store roughly one third of the world's soil carbon. Peatlands also comprise a large natural source of methane emitted to the atmosphere. Some methane in peatlands is oxidized before escaping to the atmosphere by aerobic methane oxidizing bacteria. With changing climate conditions, the fate of the stored carbon and emitted methane from these systems is uncertain. One important step toward better understanding the effects of climate change on carbon cycling in peatlands is to ascertain the microorganisms actively involved in carbon cycling. To investigate the active aerobic methane oxidizing bacteria in a boreal peat bog, a combination of microcosm experiments, DNA-stable isotope probing, and next generation sequencing technologies were employed. Studies were conducted on samples from the S1 peat bog in the Marcell Experimental Forest (MEF). Potential rates of methane oxidation were determined to be in the range of 13.85 to 17.26 μmol CH₄ g dwt⁻¹ d⁻¹. After incubating with ¹³C-CH₄, DNA was extracted from these samples, separated into heavy and light fractions with cesium chloride gradient formation by ultracentrifugation and needle fractionation, and fractions were fingerprinted with automated ribosomal intergenic spacer analysis (ARISA) and further interrogated with qPCR. Based on ARISA, distinct banding patterns were observed in heavy fractions in comparison to the light fractions indicating an incorporation of ¹³C into the DNA of active methane oxidizers. This was further supported by a relative enrichment in the functional gene pmoA, which encodes a subunit of the particulate methane monooxygenase, in heavy fractions from samples incubated for fourteen days. Within heavy fractions for samples incubated for 8 and 14 days, the relative abundance of methanotrophs increased to 37% and 25%, respectively, from an in situ abundance of approximately 4%. Phylogenetic analysis revealed that the methanotrophic community was composed of both Alpha and Gammaproteobacterial methanotrophs of the genera Methylocystis, Methylomonas, and Methylovulum. Both Methylocystis and Methylomonas have been detected in peatlands before, however, none of the phylotypes in this study were closely related to any known cultivated members of these groups. These data are the first to implicate Methylovulum as an active methane oxidizer in peatlands, though this organism has been detected in another cold aquatic ecosystem with consistent methane emissions. The Methylovulum sequences from this study, like Methylocystis and Methylomonas, were not closely related to the only cultivated member of this genus. While Methylocystis was dominant in ¹³C-enriched fractions with a relative abundance of 30% of the microbial community after an eight-day incubation, Methylomonas became dominant with a relative abundance of approximately 16% after fourteen days of incubation. The relative abundance of Methylovulum was maintained at 2% in ¹³C- enriched fractions after eight and fourteen days.

Nitrification, a key step in biological nitrogen removal processes, is the oxidation of ammonia into nitrate performed by ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) under aerobic condition. Researchers have focused on factors affecting the performance of nitrification for decades, but the inorganic carbon limitation on nitrification had been neglected. However, the increase in nitrogen in wastewater has increased the need to evaluate and improve our understanding of this limitation. In a previous research, the hypothesis that different inorganic carbon concentrations would enrich different AOB populations has been examined. In this study, the focus was on the effect of inorganic carbon concentration on NOB, which has a close relationship with AOB. Two 5L lab–scale continuous–flow stirred tank reactors (CSTR) were operated to evaluate the nitrification performance and microbial ecology of nitrifier populations acclimated under inorganic carbon sufficient (high–IC) and limited (low–IC) conditions for approximately 700 days. During the operation period, both bioreactors were able to maintain satisfactory nitrification efficiency higher than 95% at an influent ammonium concentration of 250 mg–N/L. Nitrate was the major end product and no significant nitrite accumulation was observed. To evaluate the effects of inorganic carbon on NOB community structures, cloning/sequencing and real–time PCR were applied to target and quantify the two common NOB genera, Nitrospira and Nitrobacter, as no molecular probe targeting all known NOB is available presently. The results showed that these two genera were both found in the two reactors. Nitrospira was the dominant NOB population in the high–IC bioreactor, while Nitrobacter was dominant in the low–IC one after one year acclimation. Kinetic analysis revealed that NOB enriched in the two reactors have different kinetic performances. However, IC concentration did not show a significant impact on the nitrite oxidizing kinetics of NOB in the batch tests.

La zone latérale des filaments branchiaux des bivalves Codakia orbiculata et Lucina pensylvanica est le lieu d'une symbiose chimioautotrophe avec des bactéries sulfo-oxydantes hébergées dans des cellules spécialisées, appelées bactériocytes. Dans cette étude, nous nous sommes proposés de déterminer les mécanismes qui sous-tendent la plasticité cellulaire et tissulaire observée au cours des processus de décolonisation et de recolonisation bactérienne. Pour ce faire, des individus collectés dans leur milieu naturel, ont été maintenus au laboratoire dans des bacs d'eau de mer filtrée en absence de nourriture et de soufre réduit, afin de provoquer la décolonisation bactérienne des filaments branchiaux. Lorsque la branchie apparaissait purgée de ses symbiotes, les individus ont été remis dans leur habitat naturel afin de provoquer sa recolonisation. L'analyse des branchies au cours de ces processus a fait appel à des techniques variées (histologie, immunohistochimie, hybridation in situ, cytométrie en flux, dosage des protéines et spectrométrie de fluorescence X). Les résultats obtenus ont permis de montrer que l'acquisition environnementale mise en évidence chez les juvéniles des Codakia se poursuivait tout au long de la vie des adultes. Cette étude a également permis une meilleure compréhension des mécanismes tissulaires sous-jacents à la plasticité du filament branchial en mettant en évidence les processus d'apoptose et de prolifération cellulaire qui ont lieu au cours des processus de décolonisation et de recolonisation. Ces processus s'accompagnent d'une variation de la teneur en soufre élémentaire, ainsi que de la taille relative et du contenu génomique des symbiotes / The lateral zone of gills filaments of coastal bivalves Codakia orbiculata and Lucina pensylvanica is the site of chemoautotrophic symbiosis with sulfur-oxidizing bacteria, housed in specialized cells called bacteriocytes. The objective of this thesis is to determine the mechanisms underlying cel1 plasticity and tissue plasticity observed in the lateral zone of gills filaments during the processes of bacterial decolonization and recolonization. In order to do this, the individuals collected in their natural habitat were maintained at the laboratory in seawater filtered tanks, without food and reduced sulfur, to cause bacterial decolonization. When the gills seemed to be purged, the individuals were returned to their natural habitat in order to cause the bacterial recolonization of gills filaments. The analysis of the gills during these processes involves several techniques (histology, immunohistochemistry, molecular hybridization, flow cytometry, total protein assays, protein sulfur assays, X-ray fluorescence spectrometry).This study shows that symbiont acquisition can occur during the entire life of Codakia bivalves. It also allows a better understanding of gills filaments plasticity by highlighting apoptosis and cell proliferation during decolonization and recolonization processes.Theses processes are accompanied with of elemental sufur, relative size and genomic content of symbiontes.

Mercury (Hg) is a toxic heavy metal that poses significant human and environmental health risks. Mineral-associated Hg is the largest reservoir of Hg in the environment where it can account for nearly 60% of the global Hg mass inventory. A large fraction of this pool is comprised of mercury sulfide (HgS) minerals, including metacinnabar (beta-HgS). HgS minerals have long been considered insignificant sources of Hg to aqueous or atmospheric pools in all but severely acidic environments due to their low solubility and slow abiotic dissolution kinetics. Little previous work has been conducted investigating the bacterial colonization of HgS minerals and the potential role of these mineral-associated communities in impacting the mobility of mineral-hosted Hg. To address this gap in knowledge, the studies within this dissertation employed a combination of field- and laboratory-based methods. Using culture-independent techniques, this work revealed that sulfur-oxidizing bacteria can extensively colonize metacinnabar within aerobic, near neutral pH, creek sediments, suggesting a potential role for chemolithotrophic bacteria in metacinnabar weathering. Within laboratory incubations, the dominant bacterial colonizer (Thiobacillus thioparus), induced extensive release and volatilization of metacinnabar-hosted Hg. These findings expose a new pathway for metacinnabar dissolution and point to mineral-hosted Hg as an underappreciated source of elemental Hg that may contribute to global atmospheric Hg budgets. In addition, this work elucidates the importance of thiosulfate, a major intermediate sulfur species in the environment, in stimulating metacinnabar dissolution. Therefore, the work within this dissertation shows that authigenic HgS minerals are not merely a sink for Hg within non-acidic natural environments and instead are a source of dissolved and gaseous Hg. This work provides critical information for predicting the transport of Hg in the environment and for developing appropriate management and remediation strategies for Hg-contaminated systems. / Engineering and Applied Sciences

Syntrophic acetate oxidizing bacteria convert acetate into hydrogen and carbon dioxide and through the mutualistic syntrophic partnership with methanogens the products are further converted to methane in biogas processes operating at high ammonia concentrations. There is very little known about SAOBs, only five have been characterized and had their genome analyzed. The aim of this project was to gain further knowledge about the methane producing pathway of SAOBs with a proteomic approach. Proteins were extracted from biogas sludge with a phenol-based approach and trypsin digestion and peptide recovery were performed using the Suspension Trapping method. Measurement of the peptide content was made with LC-MS/MS. The peptide profiles obtained were screened for the proteins expressed of the mesophilic SAOB Syntrophaceticus schinkii. The data supports earlier suggestions that it utilizes the Wood-Ljungdahl pathway for hydrogen production. Furthermore, the peptide profile revealed that enzymes for the glycine reductase complex and the glycine cleavage system were expressed during high ammonia concentration, indicating a potential role of these enzymes in the methane producing pathway. However, due to partial failure of the sample preparation for mass spectrometry measurements no quantification conclusions could be made. A discussion on how to further improve sample preparation methods as well as how to access the proteome to a large extent is presented.

>Magister Scientiae - MSc / Molecular methods were used to investigate the microbial diversity and community structure of ammonia-oxidizing bacteria (AOB) associated with the roots of the Proteaceae plant family. The identification of ammonia oxidizing bacteria in this ecosystem is of particular interest since Proteaceae are adapted to acidic, low nutrient (e.g. nitrogen) soils. The ammonia monooxygenase operon was used as a molecular marker to identify ammonia-oxidizing bacteria associated with the proteoid roots of the three Proteaceae members and compared to non-plant associated soil. PCR amplification using primer sets targeting the ammonia monooxygenase gene (amoA subunits) were used to construct a clone library. Sequence diversity was determined by RFLP analysis of amoA to identify major groups of AOB of the ~-subclass of Proteobacteria in total community DNA, and DNA sequencing and phylogenetic analysis were also applied. DGGE analysis was performed to determine the community structure and distribution of ammonia-oxidizing bacteria in plant-associated and non-plant associated soils. The AOB genotypic diversity was similar in the plant-associated samples and non-plant associated soil. All AOB phylotypes belonged to Nitrosospira species and clustered with Nitrosospira cluster 3. The abundance of the amoA was quantified to be approximately 4.2 x 107 copies/g of dry soil, using a real-time PCR assay. These data suggest that the Nitrosospira species are the dominant phylotypes in that environment. This investigation provides new insights into the relationships between plants and ammonia-oxidizing bacteria in natural Fynbos ecosystems.

Ammonia-oxidizing bacteria (AOB) are chemolithotrophs that oxidize ammonia/ammonium to nitrite in a two-step process to obtain energy for survival. AOB are difficult to isolate from the environment and iso lated strains may not represent the diversity in soil. A genetic database and molecular tools were developed based on the ammonia monooxygenase (AMO) encoding genes that can be used to assess the diversity of AOB that exist in soil and aquatic environments without the isolation of pure cultures. The amo genes have excellent potential as molecular markers; since AMO is only found in the AOB and is essential for their metabolism, AOB must carry at least one functional copy of the amo operon. The operon is composed of at least three genes, amoC, amoA. and amoB (encoding for the subunits AmoC, AmoA, and AmoB). The amoC gene was first discovered and its sequence was obtained from Nitrosospira sp. NpA V. The amooperon is found in several copies within AOB genomes in the β-subdivision but as a single copy in y-subdivision genomes. In Southern analysis, cross-hybridization was only observed between amo genes within a subdivision. They-subdivision amo sequences have higher identity values to the genes encoding the related particulate methane monooxygenase than to the β-subdivision amo sequences. Since amoA encodes the subunit containing the active site, it was sequenced entirely for all the strains studied (16 amoA sequences total). The amoC and amoB genes were also sequenced for several strains. The amo genes allow for better discrimination between closely related strains than the 16S rRNA genes. In all cases, the amo operon consists of amoC, followed by a variable length intergenic region, and then by amoAB. The variability in length of the intergenic region is strain specific, and is therefore potentially useful for profiling AOB communities. The amo-gene database was the basis for the design of conserved oligonucleotide primers for the polymerase chain reaction (PCR). These primers were used to amplify amo sequences from a mixed template of DNA extracted directly from soil. Results indicate that the amo genes are excellent molecular markers for the assessment of AOB communities in the environment.

No description available.