Abstract The process of nitrification is highly dependent on the microbial activities and transformation, which is carried out by autotrophic nitrifiers in general, however some heterotrophic nitrifiers also can carry out the process. The diversity of autotrophic and heterotrophic ammonia-oxidizing bacteria in aquaculture ponds in Kaohsiung county was investigated. Ten heterotrophic bacteria were isolated. The nitrification ability and 16S rDNA sequences were determined. Seven of the strains had higher nitrification ability, five of them are belong to the genus of Pseudomonas, and the other two belong to Alcaligenes and Serratia, respectively. Both 16S rDNA and amoA gene sequences results showed that all autotrophic ammonia-oxidizing strains in this study belong to Nitrosomonas genus. From the data of 16S rDNA sequences, the strains isolated from Linyuan Shiang were distinct to the other two sites. Besides, amoA gene represents a very powerful molecular tool for analyzing ammonia-oxidizing bacteria communities due to its specificity and fine-scale resolution of closely related populations.

Floodplain lakes are wetlands which receive flood waters from nearby rivers or other sources. Water samples were taken from floodplain lakes near the Illinois River, the Mississippi River, and the Cache River in Southern Illinois. Fluorescence in situ hybridization (FISH), spectrophotometry, and gene probes were used to investigate the effect of nutrient and chemical concentrations on the abundance of nitrifying bacteria; specifically ammonia-oxidizing Nitrosococcus and Nitrosomonadales and nitrite-oxidizing Nitrospira and Nitrobacter. Nitrosococcus was the dominant ammonia-oxidizing bacteria at each river system. Nitrospira and Nitrobacter had similar average abundances. Nitrosococcus abundances showed a significant positive correlation with nitrate (NO3-) (R2= 0.247, P=0.05, 95% confidence R2≥0.199) and a positive trend with nitrite (NO2-) (R2= 0.194, P=0.10, 90% confidence R2≥0.125). Nitrosomonadales abundance positively correlated with temperature (R2= 0.530, P=0.05, 95% confidence R2≥0.510). Nitrospira abundances positively correlated with ammonium (NH4+) (R2= 0.265, P=0.05, 95% confidence R2≥0.199), NO2- (R2= 0.372, P=0.05, 95% confidence R2≥0.199), and NO3- (R2= 0.482, P=0.05, 95% confidence R2≥0.199). None of the target bacterial abundances significantly correlated with pH or dissolved inorganic phosphate.

>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.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20009号 / 農博第2193号 / 新制||農||1045(附属図書館) / 学位論文||H28||N5018(農学部図書室) / 33105 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Due to the adverse effects of ammonia on the environment, many governments, including Canada, have imposed new regulations to reduce the discharge of ammonia wastewater effluent into natural receiving waters, which has resulted in the upgrade of ammonia removal at water resource recovery facilities (WRRFs) across the world. There is therefore a need to investigate present urban/peri-urban and rural challenges associated with municipal total ammonia (TAN) removal. In particular, there is a need to further advance and optimize technologies such as the moving bed biofilm reactor (MBBR) to meet these critical challenges. The first objective of this thesis is to validate an elevated loaded strategy for partial nitritation (PN) MBBR as an application for mainstream urban and peri-urban municipal wastewater treatment and to elucidate the mechanism of nitrite-oxidation suppression of this system. The second objective is to identify practical storage strategies for nitrifying MBBR units as rural municipal wastewater upgrade systems (lagoon systems), optimizing the TAN removal performance during seasonal discharge periods. In the context of the present climate change crisis and sustainable development requirements, there is an increased need for efficient TAN removal from urban and peri-urban municipal wastewaters. The application of the energy and cost-efficient partial nitritation/anammox (PN/A) technology to mainstream urban and peri-urban municipal wastewater can prove challenging because of limited ability to achieve the stable PN. Hence, there is a need for the validation of the present strategies for achieving effective and stable PN in the mainstream portion of conventional urban and peri urban WRRFs. The 45 days operation of a laboratory-scale, elevated loaded PN MBBR with average surface area loading rate (SALR) of 5.2 ± 0.1 g TAN/m²·d and a hydraulic retention time of 2h showed a successful and stable nitrite accumulation. The average surface area removal rate (SARR) of 2.3 ± 0.2 g TAN/m²·d (theoretical performance objective of 2.7 g TAN/m²·d), TAN removal efficiency of 43.1 ± 3.4% (theoretical performance objective of 53%) and NO₂- / (NO₂- + NO₃-) ratio of 82.4 ± 4.8% (theoretical performance objective of 100%) meets the necessary requirement to support subsequent cost-efficient anammox process. Biofilm analyses of the laboratory-scale, elevated loaded PN MBBR indicated that the attached biofilm was thick and dense, stable biofilm that did not show and biofilm loss or washout. Biofilm cell viability analyses was indicative of an active biofilm. The ratio of AmoA gene targets of the ammonia oxidizing bacteria (AOB) in the MBBR biofilm to the targeted gene region of the Nitrospira nitrite oxidizing bacteria (NOB) population demonstrates that NOB activity suppression of this technology was the dominant mechanism of nitrite-oxidation in the elevated loaded PN MBBR system. In North America, the TAN removal performance of waste stabilization ponds (also termed wastewater treatment lagoon systems), which are widely applied as rural WRRFs, is often not stable due to seasonal temperature variations. Nitrifying MBBR as an upgrade TAN removal unit has been successfully applied to improve TAN removal during winter. However, re-seeding the nitrifying MBBR biofilm during each seasonal operation period is not sustainable. There is therefore an urgent need for optimizing storage strategies of nitrifying MBBR carriers when used as TAN removal upgrade systems of rural WRRFs. The study of storage strategies for nitrifying MBBR as lagoon upgrading systems indicated the batch storage of the nitrifying MBBR biofilms with intermittent aeration could be an effective storage strategy for short-term (12 weeks) storage. Carriers stored in continuous flow aerated condition was shown to be the second most suitable storage method for nitrifying MBBR carriers for systems exposed to less than 12 weeks of storage. Carriers stored in dry condition, batch aerated conditions without flow, and continuous flow aerated condition for long-term (over 18 weeks) failed to achieve full nitrification following 18 days of operation conditions. Carriers stored in dry condition did not successfully achieve full nitrification for short-term and long-term storage and may not be applied to store full nitrification MBBR carriers. The study suggested that, compared to re-seeding start up strategy of the lagoon upgrading nitrifying MBBR biofilm, the use of the appropriate storage strategies, such as batch aerated conditions without flow, has the potential to shorten the start-up time and save energy during the non-discharge periods.

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Understanding how nitrifiers react when exposed to low DO conditions could provide a greater understanding of low DO operations in full-scale biological wastewater treatment. Previous methods to observe nitrifier oxygen kinetics do exist in literature, however they are inefficient and labor intensive. Other more efficient methods require the use of selective inhibitors, which alter the characteristics of the biomass. This study developed a time and labor efficient respirometric method to distinctly measure oxygen half-saturation coefficients for both ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) without the use of selective inhibitors. By eliminating the use of inhibitory substances, representative biomass characteristics were maintained throughout the tests. The developed method, called the declining DO method, consisted of using a high-speed dissolved oxygen (DO) probe to measure relative oxygen uptake rates (OUR) within a batch reactor when varying substrates (ammonia and nitrite) were present in excess within the system. A forward model was developed based on Monod kinetics to simultaneously fit Monod curves to the experimental OUR data. These curves were fit by solving for optimum oxygen kinetic parameters representing endogenous respiration, NOB, and AOB. An inverse model using Markov chain Monte Carlo analysis was applied to the results found in the forward model to provide statistical validation of the proposed respirometric method. A separate method, called the substrate utilization rate test, was conducted in parallel with the declining DO tests to compare and verify oxygen half-saturation coefficient results. Parallel tests were conducted using biomass samples from three different Hampton Roads Sanitation District (HRSD) full-scale facilities. Operating conditions between the three HRSD facilities were considered when performing parallel testing, including averages for DO, solids retention time (SRT), and floc size. Average floc size was found to have a significant effect on the observed oxygen half-saturation values. Observed trends for the KO values estimated using the two methods remained consistent throughout all tests, where KO,NOB was always lower than KO,AOB. The comparison of the two methods highlighted some faults associated with the substrate utilization rate test, which is commonly used in literature to observe nitrifier oxygen kinetics. The declining DO method appeared to be more resistant to potential experimental error and required less than half the time compared to the substrate utilization rate test. The development of the declining DO method without the use of selective inhibitors provided a more time and labor efficient technique for estimating apparent KO values for NOB and AOB without sacrificing biomass characteristics representative of the full-scale treatment process. Biomass samples collected from variable treatment process conditions yielded consistent parallel test results, providing further evidence that the proposed declining DO method can be a robust and reliable technique for distinctly measuring apparent oxygen half-saturation values for NOB and AOB. / Master of Science / Wastewater treatment operations utilizing biological nitrogen removal (BNR) require a continuous supply of oxygen for aerobic processes. Energy costs associated with aeration generally accounts for at least 50% of the total energy consumption at conventional activated sludge wastewater treatment facilities. Operating aerobic zones at low average dissolved oxygen (DO) concentrations could be an effective way to significantly reduce aeration costs as well as material costs associated with BNR treatment processes. This study developed a method to measure oxygen kinetics for the two groups of autotrophic bacteria responsible for performing nitrogen removal. The method consisted of measuring relative oxygen uptake rates (OUR) within a batch reactor when varying substrates were available. This method is unique from previously developed techniques in that the use of selective inhibitors was not included, meaning the characteristics of the wastewater were largely unchanged and therefore better represent biomass conditions within the full-scale process. The results of the proposed method were verified using an alternate method for estimating oxygen kinetics. These two methods were conducted in parallel using biomass samples from several full-scale Hampton Roads Sanitation District wastewater treatment facilities utilizing a variety of process designs and operating conditions. Consistent results obtained between the two methods suggested the proposed method is an effective technique for distinctly measuring nitrifier oxygen kinetics.

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