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.

Soluble manganese (Mn) presents a significant treatment challenge to many water utilities, causing aesthetic and operational concerns. Surface water treatment plants using ozonation followed by biofiltration are unable to apply free chlorine across the filter, a method used by many utilities for soluble Mn control. These facilities are vulnerable to periodic problems of elevated Mn in the finished water. Manganese-oxidizing bacteria (MOB) readily oxidize Mn in groundwater treatment applications, which normally involve pH values above 7.0.  However, the ability of MOB to facilitate Mn oxidation under lower pH conditions (6.2--6.3) often employed to optimize organics removal has not been demonstrated. Laboratory-scale biofilters were operated to evaluate the ability of MOB to accomplish soluble Mn control at a range of pH (6.3--7.3).  The biofilters were able to oxidize Mn at a pH as low as 6.3 at greater than 98% removal.  Ozonation by-product removal was also greater than 90% in all filter columns. Stress studies indicated that well-acclimated MOB can withstand variations in Mn concentration, hydraulic loading rate, and temperature typically found at surface water treatment plants, at least for relatively short (1-2 days) periods of time. Pilot studies demonstrated that there are unknown factors that affect MOB acclimation, and MOB may be present in full-scale biofilters already. MOB are difficult to identify with current biological analysis techniques and comprise small percentages of the total microbial population. MOB have demonstrated potential for use in surface water treatment plants, but further research is needed before this application is fully feasible. / Master of Science

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.

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.

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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Deammonification is a widely used technology for side stream treatment with rich ammonium streams at relatively high temperatures, such as, the reject water coming from dewatering units in treatment of digested sludge and industrial wastewaters. The deammonification process has lower operational costs than conventional systems, consumes less energy, enables the increase of biogas production and it is easy to implement. However, this technology has not yet been applied in full- scale mainstream treatment due to its restrictions in coping with high C/N ratios, low temperatures, and the need for post-treatment processes. These conditions are allegedly negative to the growth and performance of anammox bacteria affecting the bacterial groups’ behavior in the process. This master thesis project aimed to evaluate the feasibility of using deammonification to remove nitrogen from mainstream wastewater, which was studied by monitoring the bacterial activity in a pilot scale reactor. The different bacterial groups involved (AOB, NOB, heterotrophs, and denitrifiers) were monitored by weekly measuring their activity in batch activity tests. The results allowed the evaluation of different operational scenarios and their impact by following up on the changes in the bacterial competition. The study was conducted for six months in a single-stage IFAS (integrated fixed-film activated sludge) pilot-scale reactor located in Stockholm and fed with pretreated (with a UASB) municipal wastewater. The different operational scenarios involved changes in temperature, aeration patterns, DO concentration, SRT, and HRT. The adjustment of these features was done in the interest of promoting AOB and anammox bacterial growth, leading to an improvement of the deammonification efficiency in future studies. However, the chosen operational conditions were to enhance bacterial competition and facilitate its visualization, not to maximize nitrogen removal. Thus, the most suitable scenario found during this study included DO concentration of 1.5 mg/L with 10 aeration-20 non-aeration pattern and ensured nitrogen removal rates within normal values while allowing the monitoring of all the bacterial groups. TN removal reached a value above 50% and NH4-N above 95%, whereas nitrogen Removal Rate (NRR) increased to 30g/N/m3-d and the system had an overall nitrogen removal efficiency of 75%. Nevertheless, it was proven that in the right environment, the necessary bacterial groups can be selectively accumulated and successfully perform deammonification and reduce nitrogen levels in mainstream wastewater. / Deammonifikation är en välanvänd teknik för rening av sidoströmmar med höga ammoniumkoncentrationer vid relativt hög temperatur, som till exempel rejektvatten från avvattning av rötslam eller industriellt avloppsvatten. Deammonifikationsprocessen har lägre driftkostnad än konventionella reningsprocesser, förbrukar mindre energi samt möjliggör högre biogasproduktion samtidigt som processen är enkel att implementera. Reningstekniken har dock ännu inte tillämpats i fullskala för rening av huvudströmmen på grund av den höga C/N-kvoten och de låga vattentemperaturerna i kommunalt avloppsvatten samt behovet av efterbehandling. Detta anses ha en negativ inverkan på anammoxbakteriernas tillväxthastighet och funktion vilket påverkar bakteriegruppens beteende i processen. Syftet med detta examensarbete var att utvärdera om det är praktiskt genomförbart att använda deammonifikation för att rena kväve från kommunalt avloppsvatten, vilket följdes upp genom att studera bakterieaktiviteten i en pilotskalereaktor. De involverade bakteriegrupperna (AOB, NOB, heterotrofer och denitrifierare) övervakades genom att mäta den mikrobiella aktiviteten varje vecka med hjälp av batch-tester. Resultaten användes till att utvärdera olika driftstrategier och deras effekt genom att följa förändringarna i mikrobiell aktivitet hos de konkurrerande bakteriegrupperna. Studien genomfördes i Stockholm under sex månader i en enstegs-IFAS-pilotskalereaktor (integrerad process med biofilm på fast bärarmaterial och aktivslam) som matades med kommunalt avloppsvatten som förbehandlats i en UASB-reaktor. De olika driftstrategierna omfattade olika temperaturer, luftningsstrategier, syrekoncentrationer, slamåldrar och hydrauliska uppehållstider. Syftet med driftstrategierna var att främja AOB- och anammoxbakteriers tillväxt för att i framtida studier kunna erhålla en förbättrad deammonifikationsprocess. Syftet i denna studie var dock i första hand att förbättra den bakteriella konkurrensen och göra den lättare att mäta, inte att uppnå bästa möjliga kväverening. Den driftstrategi som gav bäst resultat i denna studie innebar att hålla en syrehalt på 1,5 mg/l med 10 minuter luftning följt av 20 minuter utan luftning vilket säkerställde en normal kväveavskiljning och samtidigt möjliggjorde övervakning av samtliga fyra bakteriegrupper. Totalkväveavskiljningen var över 50 % och ammoniumavskiljningen över 95 % medan kvävereningsaktiviteten ökade till 30 g N/m3-d och systemet hade en övergripande effektivitet på 75 %. Studien visade att under rätt förutsättningar kan de nödvändiga bakteriegrupperna selekteras fram och deammonifikation av kommunalt avloppsvatten kan utföras på ett framgångsrikt sätt.