Дисертації з теми "Bacterias anammox"

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-graduação em Ecologia, Florianópolis, 2009
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O presente estudo teve como objetivo caracterizar e analisar filogeneticamente bactérias anaeróbias oxidadoras de amônio (ANAMMOX) coletadas de bioreator de bancada de fluxo ascendente inoculado com lodo aclimatado proveniente de lagoa anaeróbia de um sistema de Lagoas de Tratamento de Dejetos de Suínos e padronizar técnica de Reação em Cadeia da Polimerase (PCR) objetivando amplificação de uma região de 436 pares de base (pb) correspondente a subunidade menor (16S rRNA) do ribossomo de bactérias ANAMMOX. Visando a identificação dos organismos presentes no bioreator, foram utilizadas as técnicas de PCR, clonagem e sequenciamento da região 16S rRNA e dos fragmentos de 436 pb amplificados. Os iniciadores desenvolvidos foram avaliados quanto sua especificidade e limite de detecção. Foram obtidos 29 clones, dos quais 17 carregavam o gene 16S rRNA e 12 carregavam o fragmento de 436 pb. Entre os 17 clones obtidos, três apresentaram 97% de identidade com ANAMMOX Candidatus Jettenia asiática e Planctomycete KSU-1, 12 tiveram identidade com Janthinobacterium (99%) e dois apresentaram similaridade com clones não cultivados. Dos clones carregando o fragmento de 436 pb, oito apresentaram 96-100% de semelhança com ANAMMOX Candidatus Anammoxoglobus propionicus, Planctomycete KSU-1 e Candidatus Jettenia asiatica. Um clone teve 99% de similaridade com Pseudomonas sp. e outros três clones apresentaram semelhança com clones não cultivados. Embora os iniciadores tenham amplificado fragmentos genômicos de organismos não-ANAMMOX, o teste de limite de detecção mostrou que com a PCR foi possível amplificar a região alvo usando concentrações extremamente baixas (0,3 ng) de material genético. A utilização de tais ferramentas (extração de material genômico e execução de PCR com os novos iniciadores aqui desenvolvidos) mostrou-se eficiente, econômica e de fácil execução para caracterização de organismos ANAMMOX, abrindo uma gama de oportunidades para ampliar nosso conhecimento sobre estas bactérias e consequentemente melhorar o tratamento de dejeto de suíno.

Pharmaceutically active compounds (PhACs) and endocrine disrupting compounds (EDCs) can pose a significant risk to the environment and human health, undermining prosperity. Current wastewater treatment plants (WWTPs) cannot efficiently act as barriers to their release and have been identified as main points of discharge and contamination. The present thesis aimed to investigate the fate of five PhACs (ibuprofen, sulfamethoxazole, metoprolol, carbamazepine and venlafaxine) and five EDCs (estrone, 17β-estradiol, estriol, 17α-ethinylestradiol and bisphenol A) in different systems simulating wastewater treatment scenarios and to identify factors triggering their elimination. A comparative assessment was carried out to determine the contribution of the microbial groups (either autotrophic or heterotrophic) present in different lab, pilot and full-scale treatment systems performing different processes in the removal of the selected compounds. The results indicated that the overall efficiency of wastewater treatment systems can be broadened by combining different aerobic and anaerobic conditions and different types of biomass
Els compostos farmacèuticament actius (PhACs) i els pertorbadors endocrins(EDC) poden suposar un risc considerable per al medi ambient i la salut humana. Les estacions depuradores d'aigües residuals (EDAR) no poden actuar de manera eficient com a barreres per al seu alliberament i s'han identificat com a punts principals de descàrrega. La present tesi pretén determinar el destí de cinc PhACs (ibuprofèn, sulfametoxazol, metoprolol, carbamazepina i venlafaxina) i cinc EDCs (estrona, 17β-estradiol, estriol, 17α-etinilestradiol i bisfenol A), en sistemes que simulen escenaris de tractament d'aigües residuals, per identificar els factors claus en la seva eliminació. Es va realitzar una avaluació comparativa per determinar la contribució dels diferents grups bacterians (autòtrofs o heteròtrofs) presents en diferents sistemes a escala de laboratori, pilot i a gran escala. Els resultats indiquen que l'eficiència global dels sistemes de tractament d'aigües residuals es pot ampliar combinant diferents condicions aeròbiques i anaeròbies i tipus de biomassa

This thesis results from the collaborative projects between the LEQUIA-UdG group and Cespa (a company in charge of several landfill sites in Spain). The aim of the work was the development of a suitable alternative treatment for nitrogen removal from mature landfill leachates. The thesis presents the application of the anammox (anaerobic ammonium oxidation process) process to treat ammonium rich leachates as the second step of the PANAMMOX® process. The work deals with preliminary studies about the characteristics of the anammox process in a SBR, with special focus on the response of the biomass to nitrite exposure. The application of the anammox process with leachate was first studied in a lab-scale reactor, to test the effect of the leachate matrix on anammox biomass and its progressive adaptation. Finally, a start-up strategy is developed and applied for the successful start-up of a 400L anammox SBR in less than 6 months.
Aquesta tesi és fruit de la col•laboració entre el grup LEQUIA-UdG i Cespa. L'objectiu del treball va ser el desenvolupament d'un tractament alternatiu per a l'eliminació biològica de nitrogen dels lixiviats madurs d'abocador. La tesi presenta l'aplicació del procés anammox (anaerobic ammonium oxidation) per tractar elevades càrregues de nitrogen en el segon pas del procés PANAMMOX ®. El treball inclou estudis preliminars sobre les característiques del procés de anammox en un SBR, amb especial atenció a la resposta de la biomassa a l'exposició de nitrit. L'aplicació del procés anammox amb lixiviat es va estudiar inicialment en un reactor a escala de laboratori, per provar l'efecte de la matriu del lixiviat sobre la biomassa anammox i la seva adaptació progressiva. Finalment, es va desenvolupar una estratègia de posada en marxa que va ser aplicada amb èxit per a la posada en marxa d'un SBR anammox de 400L en menys de 6 mesos.

Partial Nitritation/Anammox process (deammonification process), by which occurs oxidation of ammonium to nitrogen gas by autotrophic bacteria in anaerobic conditions, considered to be cost-effective and environmentally friendly method of nitrogen removal. Present research work focuses on achieving a high nitrogen removal degree, thanks to Anammox bacteria, while providing the best performance of the ongoing process. Integrated fixed-film activated sludge (IFAS) reactor was supplied with the main stream of the wastewater after UASB reactor, characterized by low concentration of nitrogen and organic matter. The bacteria ability to accommodate, were tested in the biofilm and in the activated sludge, depending on the different stages in which the process were being conducted. Batch test, such as Specific Anammox Activity (SAA), Nitrate Uptake Rate (NUR) and Oxygen Uptake Rate (OUR), were used for the evaluation of activity of various groups of bacteria. On the basis of laboratory analysis verified the values obtained from the batch tests. It was determined that a high degree of nitrogen removal (92% of NH4-N) was achieved thanks to the dominant activity of the Anammox bacteria, with low participation of other groups of bacteria. It was also proved, that Anammox bacteria activity were overwhelming in the biofilm. Dominant role of Ammonium Oxidizing Bacteria (AOB) was associated with high activity of Anammox bacteria, which together satisfyingly out-competed Nitrite Oxidizing Bacteria (NOB) and heterotrophic bacteria. It has been shown that Anammox bacteria quickly adapt to the new conditions and they are able to assume a dominant role, even in the case of inoculation of the reactor with the sludge from SBR. This allows conclude, that in the case of operational problems, the reactor can be supplied from another source, in order not to inhibit the process.

Anammox (anaerobic ammonium oxidation) is an environmentally significant process with great importance for global biogeochemical cycles. This process is mediated by a unique suite of phylogenetically distinct chemolithoautotrophic bacteria which demonstrate novel physiological and metabolic characteristics. However, despite the importance of these organisms, there is still much which is poorly understood about them, specifically the diversity and distribution of these bacteria and their controlling environmental factors. Furthermore, genomic studies and observations from the field suggest that anammox bacteria may have a far greater metabolic diversity than previously thought, suggesting that the current understanding of these organisms is incomplete. This study aimed to elucidate these aspects of the ecology of anammox bacteria in estuarine and OMZ (oxygen minimum zone) environments. A clear community shift was observed in estuarine environments from Ca. Brocadia spp. dominated freshwater sites to Ca. Scalindua spp. dominated marine sites. The OMZ was dominated by Ca. Scalindua spp. though diversity within this clade was observed between organisms in the upper oxycline and those within the core of the OMZ. Microcosm experiments amended with organic substrates suggested that some anammox organisms (namely Ca. Brocadia spp., Ca. Jettenia spp. and Ca. Kuenenia spp.) may have the ability to assimilate carbon directly from organic substrates such as dimethylamine and urea. However, these data were inconclusive and further investigations are required to prove or disprove the hypothesis that anammox bacteria can utilise organic substrates. Nevertheless, this study improves the understanding of the ecology of anammox organisms in estuarine and OMZ environments, providing an unprecedented depth of data as to the diversity and distribution and unique insights into potentially novel metabolic capabilities of these organisms.

Anammox-based processes have been commonly applied for the treatment of reject water (high nitrogen content and low C:N ratio), in the sidestream line of urban wastewater tretament plants (WWTP). However, in recent years, it has increased the interest in applying anammox-based processes in the mainstream line (lower nitrogen content), which accounts for the majority of the influent nitrogen to the WWTP. But the application of autotrophic nitrogen removal by PNA has some challenges to deal, such as low anammox growth rate, mainly at low-temperature conditions, and the suppression of nitratation process. This thesis aimed at studying suitable operational strategies to deal with the challenges aforementioned, in order to achieve stable PNA at mainstream urban WWTP. The main strategies investigated were (i) extremely low dissolved oxygen (DO); (ii) effects of temperature; (iv) influence of different IC:N ratios; and (iv) different reactors configurations: sequencing batch (SBR) and plug-flow reactor
Els processos basats en la tecnologia anammox s'han aplicat habitualment per al tractament de l’aigua de retorn de centrígues (major contingut de nitrogen i baixa relació C: N) a la línia de fangs de les estacions depuradores de aigües residuals (EDARs). No obstant això, en els darrers anys, ha augmentat l'interès en aplicar processos basats en anammox en la línia principal de l'aigua (menor contingut de nitrogen), que representa la majoria del nitrogen que es aportat a una EDAR. Però l’aplicació de l’eliminació autotrófic de nitrogen té alguns reptes a tractar, com la baixa taxa de creixement de les bacteris anammox, principalment en condicions de baixa temperatura i la supressió del procés de nitratació. Aquesta tesi té com a objectiu estudiar estratègies operatives adequades per fer front als reptes esmentats, per tal d’aconseguir un PNA estable a les principals EDARs urbanes. Les principals estratègies investigades van ser (i) oxigen dissolt (DO) extremadament baix; (ii) efectes de la temperatura; (iv) influència de diferents ràtios IC: N; i (iv) diferents configuracions de reactors: discontinu seqüencial (SBR) i de flux de pistó

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第16132号
農博第1868号
新制||農||990(附属図書館)
学位論文||H23||N4602(農学部図書室)
28711
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 左子 芳彦, 教授 藤原 建紀, 教授 澤山 茂樹
学位規則第4条第1項該当

An alternative to conventional removal of nitrogen through autotrophic nitrification and heterotrophic denitrification is autotrophic nitritation-anammox. The anammox bacteria oxidise ammonium directly to nitrogen gas with nitrite as an electron acceptor. Total autotrophic removal of nitrogen in the mainstream would bring wastewater treatment plants closer to being energy self-sufficient as it would allow for a significant reduction of aeration and an increased chemical oxygen demand reduction in the pre-treatment. An increased chemical oxygen demand reduction by mechanical treatment would potentially generate a greater biogas yield in the subsequent anaerobic digestion of the sludge. Nitritation-anammox processes have been successfully implemented over the world for treatment of ammonium rich sludge liquor of higher temperatures, while the feasibility of a mainstream implementation is still under evaluation. Lower ammonium concentrations, lower operating temperatures and better effluent quality represent the main challenges considering this energy autarkic treatment technique. Terminating nitrification at nitritation, i.e. favouring ammonia-oxidising bacteria while supressing nitrite-oxidising bacteria, is vital for a functioning nitritation-anammox process. This study aims to evaluate the suppressive effect of intermittent aeration on nitrite- oxidising bacteria while sustaining anammox activity by ex-situ batch tests in a pilot-scale moving bed biofilm reactor at Sjölunda Wastewater Treatment Plant in Malmö, Sweden. The pilot plant consists of one reactor treating sludge liquor and two mainstream reactors, connected in series, receiving effluent from a high-loaded activated sludge plant. The batch test showed a slight decrease of nitrite-oxidising bacteria activity when the reactors were intermittently aerated. Some loss in activity is expected as oxygen supply is decreased when aeration is switched from continuous to intermittent. Furthermore, the decrease coincided with an increased organic carbon loading favouring fast growing heterotrophic bacteria. The decrease in nitrite-oxidising bacteria activity can thereby be coupled with an increased competition for dissolved oxygen and space with heterotrophic bacteria. The suppression of nitrite-oxidising bacteria was not selective as results indicate a decrease in ammonia-oxidising bacteria activity as well. The nitrogen removal rate was decreased during the study while the potential anammox activity was stable in the mainstream and increased in the sludge liquor reactor. This indicates that the anammox bacteria are not hampered but rather that the availability of nitrite, i.e. the activity of ammonia-oxidising bacteria, is the limiting factor of the process.
Ett alternativ till konventionell kväverening via autotrof nitrifikation och heterotrof denitrifikation är autotrof nitritation-anammox. Anammoxbakterien oxiderar ammonium direkt till kvävgas med nitrit som elektronacceptor. Fullständigt autotrof kväverening skulle föra avloppsreningsverk närmare ett självförsörjande energiläge då luftningsbehovet minskas signifikant och en ökad reduktion av organiskt kol via mekanisk rening skulle möjliggöras. Den ökade reduktionen av organiskt kol ger potentiellt en ökad biogasproduktion i den efterkommande anaeroba rötningen av slammet. Framgångsrika nitritation-anammoxprocesser har implementerats över världen för behandling av ammoniumrikt rejektvatten med högre temperatur medan möjligheten för en huvudströmsimplementation utreds. Lägre ammoniumkoncentrationer, lägre drift- temperaturer och höga krav på utgående vattens kvalitet utgör de största utmaningarna för denna reningsteknik. Att avbryta nitrifikation vid nitritation, det vill säga gynna ammoniakoxiderande bakterier och hämma nitritoxiderande bakterier är vitalt för en fungerande nitritation- anammoxprocess. Denna studie ämnar att utvärdera den hämmande effekten av intermittent luftning på nitritoxiderande bakterier samtidigt som anammoxaktiviteten bibehålls. Detta gjordes med hjälp av ex situ -aktivitetstest med bärare från en bioreaktor i pilotskala med rörligt bärarmaterial på Sjölunda Avloppsreningsverk i Malmö. Pilotanläggningen består av en reaktor för behandling av rejektvatten och två huvudströmsreaktorer, kopplade i serie, som mottar vatten från Sjölundas högbelastade aktivslamanläggning. Aktivitetstesterna visade att aktiviteten av nitritoxiderande bakterier sjönk något. En viss minskning i aktiviteten är dock förväntad enbart utifrån att tillförseln av syre minskat då luftningsstrategin ändrats från kontinuerlig till intermittent. Minskningen av aktiviteten sammanföll även med en ökad belastning av organiskt kol, vilket gynnar snabbväxande heterotrofer. Den minskade aktiviteten av nitritoxiderande bakterier kan därmed förklaras av en ökad konkurrens med heterotrofa bakterier om löst syre och plats. De nitritoxiderande bakterierna hämmades inte selektivt då resultaten tyder på att det även skett en minskning av de ammoniakoxiderande bakteriernas aktivitet. Kväverenings- hastigheten har gått ned under studien medan den potentiella anammoxaktiviteten har varit stabil i huvudströmsreaktorerna och har ökat i rejektvattenreaktorn. Detta indikerar att anammoxbakterierna inte blivit hämmade utan att det snarare är tillgången på nitrit, det vill säga aktiviteten av ammoniakoxiderande bakterier, som är begränsande för processen.

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.

The anaerobic oxidation of ammonium (anammox) with nitrite as electron acceptor is a microbial process that generates nitrogen gas as main final product. After being discovered in the Netherlands in the 1990s, anammox has been applied in state-of-the-art biotechnologies for the removal of N pollution from ammonium rich wastewaters. The anammox process offers significant advantages over traditional nitrification-denitrification based processes. Since anammox does not need elemental oxygen, it allows for important savings in aeration. Furthermore, due to the autotrophic nature of the bacteria, anammox does not require external addition of electron donor, often needed in systems with post-denitrification. Although the anammox bacteria have high specific activity, they are slow growing, with doubling times that can range from 10 to 25 d. Therefore, in case of a toxic event causing the death of the biomass, a long recovery period will be required to reestablish full treatment capacity. The purpose of this work is to investigate the inhibition of anammox bacteria by compounds commonly found in wastewaters, including substrates, intermediates and products of the anammox reaction. Among common wastewater constituents, sulfide was shown to be especially harmful, causing complete inhibition of anammox activity at concentrations as low as 11 mg H₂S L⁻¹. Dissolved oxygen was moderately toxic with a 50% inhibiting concentration of 2.3 and 3.8 mg L⁻¹ to granular and suspended anammox cultures, respectively. Among the various compounds involved in the anammox reaction, special attention was paid to nitrite. Numerous literature reports have indicated inhibition of anammox bacteria by its terminal electron acceptor. However to date, there is no consensus explanation as to the mechanism of nitrite inhibition nor on how the inhibition is impacted by variations in the physiological status of anammox cells. The mechanisms of anammox inhibition by nitrite were thoroughly investigated in batch and continuous experiments of this dissertation. The results of this work demonstrate that conditions hindering generation of metabolic energy have a detrimental effect on the tolerance of anammox cells to toxic levels of nitrite. The absence of ammonium during events of nitrite exposure was shown to exacerbate its toxic effect. As a result of nitrite inhibition, nitric oxide, an intermediate of the anammox reaction, accumulated in the head space of the batch experiments. Moreover, nitrite inhibition was enhanced at the lowest range of pH tested (6.4-7.2), while same nitrite concentrations caused no inhibition under mildly alkaline conditions (7.5-7.8). Although other authors have relied on the classic concept that undissociated nitrous acid is the species responsible for the inhibition, the results in this work indicate that the pH affects the inhibitory effect of nitrite, irrespective of the free nitrous acid concentration. Nitrite stress triggered an active response of the anammox bacteria, which temporarily increased their ATP content to mitigate the inhibition. Additionally, starvation of anammox microorganisms, caused during storage or by sustained underloading of bioreactors, was found to limit the capacity of the bacteria to tolerate exposure to nitrite. The results of this dissertation indicate that the tolerance of anammox bacteria to NO₂⁻ inhibition relies on limiting its accumulation in sensitive regions of the cell. Active metabolism in presence of NH₄⁺ allows for active consumption of NO₂⁻, avoiding accumulation of toxic intracellular NO₂⁻ concentrations. Furthermore, secondary active transport proteins may be used by anammox bacteria to translocate nitrite to non-sensitive compartments. Nitrite active transport relies on a proton motive force. Therefore, conditions such as low pH (below 7.4) or absence of energy sources, which may disturb the maintenance of the intracellular proton gradient, will increase the sensitivity of anammox cells to NO₂⁻ inhibition. Strategies for the operation and control of anammox bioreactors must be designed to avoid exposure of the biomass to nitrite under the absence of ammonium, low pH or after periods of starvation.

Nowadays, the combination of the autotrophic nitrification together with heterotrophic denitrification is the most common process for N-removal in the WWTPs. However, when the C:N ratio is low, this two-step process does not properly work and an external C source must be added. The anammox bacteria are the target of this thesis, capable to oxidize ammonium under chemolithotrophic and anaerobic conditions. The main aim of this doctoral thesis is the search and enrichment of the anammox bacteria for a long time period, in order to be used as inocula for an anammox bioreactor. Once identified, several molecular techniques were used to optimize their detection and made it faster, to characterize their bacterial ecological partners and to study the composition of the ammonium-oxidizing bacteria and nitrite-oxidizing bacteria populations, required to obtain a suitable ammonium concentration in the influent of the anammox reactor.
Actualment, la combinació d’una nitrificació autotròfica seguida d’una desnitrificació heterotròfica és el procés més comú d’eliminació de N en les EDAR. Tot i això, quan la relació C:N és baixa aquest procés basat en dos compartiments no funciona correctament i s’ha d’afegir un font de C externa. L’objecte d’estudi d’aquesta tesi són els bacteris anammox, un nou procés capaç d’oxidar quimiolitotròficament i de manera anaeròbica l’amoni. L’objectiu principal de la tesi va ser la seva cerca i el seu enriquiment durant un llarg període de temps, per ser utilitzats com a inòculs per un bioreactor anammox. Un cop identificats es van utilitzar tècniques moleculars per d’optimitzar la seva detecció i fer-la més ràpida, caracteritzar les poblacions microbianes que van actuar com a socis ecològics i estudiar la composició de la poblacions de bacteris oxidadors d’amoni i de nitrit necessaris per aconseguir una correcta concentració d’amoni a l’influent del reactor anammox.

Excess nutrients from nonpoint sources are an ongoing problem that is expected to worsen as population and fertilizer usage rise. Conventional centralized treatment systems are not well suited to address nonpoint source pollution. More distributed best management practices (BMPs) like constructed wetlands are a promising alternative and have been widely implemented in the US since the 1970's. Constructed wetlands are multi-functional systems that can effectively store and transform harmful contaminants using primarily natural processes. However, the removal of pollutants like nitrogen by wetlands is highly variable, likely due to a combination of factors such as plant species-specific assimilation behavior, the effects of plant communities on microbial diversity and function, and variable nitrogen inputs. In this study, the effect of plant species richness (i.e., number of plant species in a system) and seasonal nutrient loading (i.e., nitrogen fertilization) on the microbial community responsible for regulating nitrogen turnover in wetland mesocosm soils was investigated. The chip-based QuantStudio 3D digital PCR (QS3D dPCR) system was used to quantify ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), comammox, anammox, and denitrifiers. Principal component analysis (PCA) was used to identify dominant patterns in the microbial community and nitrogen species. Resampling-based analysis of variance (ANOVA) was used to assess statistical significance of any observed differences caused by nitrogen fertilization or plant species richness. Results indicated that fertilization or season, which was convolved with fertilization, was the dominant factor influencing the microbial community in the study environment (27% variance explained), as indicated by the disparate clustering of fall (fertilized) and spring (unfertilized) samples about principal component 1 (fall: negative PC1, spring: positive PC1). Because unplanted unfertilized controls sampled in November clustered within the season in which they were collected rather than with other unfertilized samples collected in May, season may have influenced microbial community shifts more than fertilization for unplanted systems. This finding should be interpreted cautiously, however, given the small number of unplanted unfertilized controls (N = 2) and the absence of similar controls in the planted systems. The most abundant bacterial groups detected in May (November) were AOB, nirK, anammox, and Nitrospira spp. NOB (AOB, anammox, Nitrospira spp. NOB, and nosZ). The effects of plant species richness were more nuanced, with greater richness significantly impacting the abundance of only a subset of bacterial groups (i.e., the nitrifying bacteria AOB, Nitrospira spp. NOB, and comammox, but not the denitrifying bacteria). Different relationships between richness and microbial abundance were observed in different seasonal nutrient loadings (i.e., interaction effects between richness and fertilization were detected for some bacterial groups).
MS
As global population continues to rise, fertilizer application is becoming more commonplace in order to meet increasing agricultural demand. Fertilizers supply nutrients like nitrogen that, in excess, can negatively affect water quality. Since conventional treatment systems are largely impractical to control such diffuse, nonpoint sources of pollution, more distributed best management practices (BMPs) like constructed wetlands are a promising alternative. Several important nitrogen transformations occur within wetlands, of which soil microbial communities have a significant influence over. For instance, nitrifying bacteria can transform ammonia into nitrate and denitrifying bacteria can transform nitrate into atmospheric nitrogen. Constructed wetlands are designed to mimic these complex, dynamic processes, and can be manipulated for more effective nitrogen pollution control. However, the removal of pollutants like nitrogen by wetlands is highly variable, likely due to a combination of factors such as plant species-specific assimilation behavior, the effects of plant communities on microbial diversity and function, and variable nitrogen inputs. In this study, the effects of plant species richness (i.e., number of plant species in a system) and seasonal nutrient loading (i.e., nitrogen fertilization) on several types of nitrifying and denitrifying bacteria in wetland mesocosm soils were investigated. Digital polymerase chain reaction (dPCR) was used to quantify bacterial abundance. Principal component analysis (PCA) was used to identify dominant patterns within the data and resampling-based analysis of variance (ANOVA) was used to assess statistical significance of any observed differences caused by fertilization, season, and/or plant species richness. Results indicated that fertilization or season, which was convolved with fertilization, wasthe dominant factor influencing the microbial community in the study environment. The effects of plant species richness were more nuanced, with greater richness significantly impacting the abundance of only a subset of bacterial groups (i.e., the nitrifying bacteria AOB, Nitrospira spp. NOB, and comammox, but not the denitrifying bacteria).

Depuis la découverte des sources hydrothermales océaniques, la diversité microbienne de cet environnement a été étudiée en utilisant des approches culturales et moléculaires. Dans cette étude la diversité microbienne d’édifices hydrothermaux prélevés des sites TAG et Lucky Strike (Dorsale Médio-atlantique) a été étudiée par la réalisation d’inventaire moléculaires et de culture d’enrichissement en continu en bioréacteur. Afin de se rapprocher au mieux des conditions environnementales, les cultures ont été réalisées en condition et en présence d’hydrogéne et de dioxide de carbone ou en utilisant un mélange fluide hydrothermal/eau de mer comme milieu de culture. L’effet de changements environnementaux tel que la température et la présence ou l’absence de soufre a également été testé. La diversité des Archaea et des Bacteria a été étudiée aussi bien dans les échantillons que dans les cultures. Les analyses des séquences du gène codant pour l’ARNrlSS ont mis en évidence des micro-organismes affiliées aux Archaeoglobales, Thermococcales, Desulfurococcales, Deferribacterales et Thermales suivant les conditions de cultures. L’effet des changements environnementaux, la température, les donneurs et accepteurs d’électrons conditionnent la composition des communautés cultivées, comme cela peut se produire dans l’environnent. Dans une deuxième partie de cette étude, nous avons étudié le rôle du métabolisme anammox dans les écosystèmes hydrothermaux. La présence et l’activité de bactéries anammox ont été montrées dans ces environnements par l’amplification du gène codant pour I’ARNr16S de micro-organismes anammox, et des mesures d’activité de production de 14N-15N à haute température
Since the discovery of oceanic hydrothermal vents, microbial diversity of this extreme environment was studied using both cultivation an molecular approaches. In this study the microbial diversity of active chimney from both TAG and Lucky Strike sites (Mid Atlantic Ridge) was investigated using both molecular survey and continuous enrichment culture in a bioreactor. To reproduce as close as possible environmental conditions the cultures were performed in oligotrophic conditions, in the presence of hydrogen and carbondioxide in the ga sweeping mix or using in situ sampled fluid/sea water mix as culture medium. The effect of environmental changes such as temperature and the absence or presence of sulphur was also investigated. Archaeal and Bacterial diversity was studied in both culture and natural samples. Through 16SrRNA gene sequences analysis of the enrichment cultures, microorganisms affiliated to ArchaeogIobales, Thermococcales, Desufurococcales, Deferribacterales and Thermales were detected depending of culture conditions. The effects of environmental changes (i. E. Modification of culture conditions: temperature, electron donors and acceptors availability, medium composition) were shown to affect the microbial community in culture, as happens in their environment. We have also investigated the role of anammox in hydrothermal settings. Evidence for the occurrence of anammox in this particular habitat was demonstrated by concurren surveys including the amplification of 16SrRNA gene sequences, ladderanes lipids analysis and measurement of a 14N-15N production in isotope pairing experiments at 60 and 85°C

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 (JO2/JNH4+) 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 JO2/JNH4+ 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.

The anaerobic ammonium oxidation (Anammox) is catalyzed by anaerobic, chemolithoattotrophic bacteria and is the last discovered microbial pathway in the biological nitrogen cycle, and a new cost-effective method for removal of ammonium from wastewater. This process has been widely studied as alternative in the removal of ammonium nitrogen from wastewater due to its efficiency, lower operating costs and environmental sustainability. This study evaluates the impact of type inoculum and operating conditions on the performance of the Anammox process and microbial structure developed in reactors . Two reactors (RI and RII) of 2L, up flow fixed bed biofilm contínuos were used . Two types of inoculum were applied: one sludge coming from wastewater treatment wetland health-built (CePTS) systems, and the second coming from the treatment of industrial effluent (REGAP / Petrobras) system. These sludges were previously selected for positive PCR results presented for the detection of Anammox bacteria. The diversity of Anammox bacteria inoculum was assessed by cloning technique. The sample of the constructed wetland (inoculated in RI) was dominated by Anammox single genus, "Candidatus" Brocadia while REGAP sludge (inoculated in RII) presented three genera of Anammox : Ca. Anammoxoglobus, Ca Kuenenia and Ca. Brocadia and with predominance of Ca. Anammoxoglobus and Ca. Brocadia . It was possible to develop, Anammox activity and biomass from selected. Results reactors monitoring showed that it was possible to develop Anammox activity and biomass from selected after a year of cultured. The average ammonium and nitrite removal efficiencies of 97% were obtained after one year of operation, based on the influent NH4 +-N and NO2 --N concentrations of 110 and 145mg L-1 for both reactors respectively. The analysis of the microbial diversity of samples collected in the reactors RI and RII, after 440 days of operation, obtained by pyrosequencing technique, demonstrated that the enriched community in the reactors showed different composition, despite similar operating conditions. But the predominant groups were similar. The dominant phyla detected in both reactors were Proteobacteria, Chloroflexi, Planctomycetes and Acidobacteria. However, the phyla Acidobacteria and GN04 were first observed in Anammox reactors, suggesting its importance in this process. Regarding the Anammox community developed after 440 days of operation of the reactors, was concluded that similar operating conditions used in enrichment, selected the same Anammox population , belonging to the genus Ca. Brocadia in both reactors, despite the differences observed in the initial inoculum. Therefore, the selected sludge were able to develop the active Anammox biomass in the reactors after cultivation and operation under anoxic and autotrophic conditions. It was also observed that inocula have impact on the composition of the microbial biomass developed in the reactors, but the operating conditions applied play an important role in the selection of Anammox bacteria community.
A oxidação anaeróbia da amônia (Anammox), realizada por bactérias anaeróbias, quimiolitoautotróficas, é a via mais recentemente descoberta sobre o ciclo biológico do nitrogênio. Esse processo tem sido amplamente estudado como alternativa na remoção de nitrogênio amoniacal de águas residuárias devido a sua eficiência, menores custos operacionais e sustentabilidade ambiental. O presente estudo avalia o impacto do tipo de inóculo e condições operacionais sobre o desempenho do processo Anammox, bem como a estrutura microbiana desenvolvidos em reatores anaeróbios. Foram utilizados dois reatores, RI e RII, de leito fixo, com fluxo ascendente e contínuo (2L). Dois tipos de inóculos foram aplicados: um lodo oriundo de sistemas de tratamento de efluente sanitário- wetland construída (CePTS); e o segundo oriundo do sistema de tratamento de efluente industrial (REGAP/ Petrobras). Esses lodos foram previamente selecionados por apresentarem resultados de PCR positivos para a detecção de bactérias Anammox. A diversidade de bactérias Anammox dos inóculos foi avaliada pela técnica de clonagem. A amostra da Wetland construída (inoculada em RI) foi dominada por único gênero Anammox, Candidatus Brocadia, enquanto o lodo REGAP (inoculado em RII) apresentou três gêneros de Anammox: Ca. Anammoxoglobus, Ca. Kuenenia e Ca. Brocadia, com predominância de Ca. Anammoxoglobus e Ca. Brocadia. Resultados do monitoramento dos reatores mostrou que foi possível desenvolver atividade e biomassa Anammox a partir dos inóculos selecionados após um ano de cultivo. A eficiência média de remoção de N- NH4 + e N-NO2 - obtida, após um ano de operação, foi de 97% para uma concentração 110 and 145mg L-1 respectivamente. A análise da diversidade microbiana das amostras coletadas nos reatores RI e RII, após 440 dias de operação, realizada pela técnica de pirosequenciamento demonstrou que a comunidade enriquecida nos reatores apresentou composição diversa. Porém os grupos predominantes foram semelhantes. Os filos dominantes detectados em ambos os reatores foram Proteobacteria, Chloroflexi, Planctomycetes e Acidobacteria. No entanto, os filos Acidobacteria e GN04 foram observados pela primeira vez em reatores Anammox, sugerindo a sua importância para este processo. Em relação à comunidade Anammox desenvolvida nos reatores, concluímos que as condições operacionais semelhantes, utilizadas no enriquecimento selecionaram a mesma população de Anammox, pertencente ao gênero Ca. Brocadia em ambos os reatores, apesar das diferenças observadas no inóculo. Portanto, os lodos selecionados foram capazes de desenvolver atividade e biomassa Anammox nos reatores anaeróbios, após cultivo e operação sob condições anóxicas e autotróficas. Verificou-se, também, que os inóculos têm impacto sobre a composição da biomassa microbiana desenvolvida nos reatores, mas as condições operacionais aplicadas desempenharam papel importante na seleção da comunidade de bactérias Anammox.

Anaerobic ammonium-oxidizing (anammox) bacteria perform an important step in the global nitrogen cycle: oxidizing ammonium and reducing nitrite to form dinitrogen gas in the absence of oxygen. Anammox bacteria from the Planctomycetes phylum have been identified in a variety of natural environments but their role in groundwater ammonium oxidation has been unclear. Recent isotope studies have suggested that anammox bacteria are likely active in ammonium attenuation at contaminated groundwater sites; however, only limited biomarker-based data confirmed their presence prior to this study. I used complimentary molecular and isotope-based methods to assess the communities of anammox performing organisms at three ammonium contaminated groundwater sites in Canada: quantitative real-time PCR (qPCR), denaturing gradient gel electrophoresis (DGGE), DNA sequencing of 16S rRNA genes (with both Sanger and Illumina technologies), and ¹⁵N-tracer incubations. DNA sequencing and qPCR results demonstrated that anammox performing organisms were present at all three contaminated sites, and that they were among the dominant bacterial community members for at least one particular site (Zorra, Ontario). In addition, anammox bacterial diversity was variable. One site possessed four of five known genera of anammox performing organisms although the dominant anammox bacteria at all sites belonged to the Candidatus Brocadia genus. Isotope data from two groundwater sites showed that denitrification and anammox occurred jointly and although denitrification was the dominant process, anammox was responsible for maxima of 18 and 36% of N₂ production at these sites. By combining molecular and isotopic results I have demonstrated the diversity, abundance and activity of these anaerobic chemolithoautotrophic bacteria; these results provide strong evidence for their important biogeochemical role in attenuating groundwater ammonium contamination.

碩士
國立交通大學
環境工程系所
106
With the continuous development of the social economy and the continuous improvement of people’s living standards, the situation of nitrogen pollution in water bodies is becoming more and more serious. As a typical representative of the biological nitrogen removal technology in the new age, Anammox process has attracted attention due to its environmental protection and energy saving features, and it plays an important role in the shortcut of nitrogen removal. However, the doubling time of Anammox bacteria is long, which has become a major obstacle to the application of the process. Immobilized cell is a type of technology that use physical or chemical methods hold cells in a specific space, without affecting their ability to absorb nutrients, and at the same time have a protective effect to maintain high activity. In this study, we used both polyvinyl alcohol (PVA) and sodium alginate (SA) to form composites materials for embedding Anammox bacteria. In the previous batch experiments, the beads were made of PVA mass concentration of 13% and a SA mass concentration of 2% achieved better removal of nitrogen has been selected, and then the beads prepared from this concentration ratio was used as the experimental group. At the same time, a non-embedded control group was set up, long-term experiments were performed in two identical sequencing batch reactors (SBR), and water quality items were analyzed together with scanning electron microscope (SEM) images. The results show that with the increase of time, the pores inside the carrier can be significantly increased from the initial 10 μm to 50-80 μm, while the microorganisms can maintain a certain activity in the embedded beads. The total nitrogen removal efficiency increased continuously with the increase of the nitrogen loading rate, which eventually reached 81 ± 1%, while the control group reached 92 ± 1%. The experimental group did not show obvious superiority. After the long-term experiment was completed, the microbial activity of the two reactors were tested. During the 18 hours experiment, the nitrogen removal effect of the experimental group was 0.18 g TN-removal/d, and the control group was 0.23 g TN-removal/d, the result was also better in the control group than in the experimental group. The physicochemical parameters such as the mass transfer coefficient and specific surface area of the blank carrier were measured, and compared with related literature. It was speculated that the poor mass transfer characteristics may be the main reason for the poor nitrogen removal effect in the experimental group. In the future, the study on the mechanism of cross-linking between the embedded carrier and the Anammox bacteria, as well as the research on the influence of different embedding conditions on the activity of Anammox bacteria, has important practical significance for the practical application of the embedding technology to the Anammox process.

Anaerobic ammonium oxidation (anammox) by anammox bacteria contributes significantly to the global nitrogen cycle and plays a major role in sustainable wastewater treatment. To date, autotrophic nitrogen removal by anammox bacteria is the most efficient and environmentally friendly process for the treatment of ammonium in wastewaters; its application can save up to 60% of the energy input, nearly 100% elimination of carbon demand and 80% decrease in excess sludge compared to conventional nitrification/denitrification process. In the anammox process, ammonium (NH4+) is directly oxidized to dinitrogen gas (N2) using intracellular electron acceptors such as nitrite (NO2–) or nitric oxide (NO). In the absence of NO2– or NO, anammox bacteria can couple formate oxidation to the reduction of metal oxides such as Fe(III) or Mn(IV). Their genomes contain homologs of Geobacter and Shewanella cytochromes involved in extracellular electron transfer (EET). However, it is still unknown whether anammox bacteria have EET capability and can couple the oxidation of NH4+ with transfer of electrons to extracellular electron acceptors. In this dissertation, I discovered by using complementary approaches that in the absence of NO2–, freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to carbon-based insoluble extracellular electron acceptors such as graphene oxide (GO) or electrodes poised at a certain potential in microbial electrolysis cells (MECs). Metagenomics, fluorescence in-situ hybridization and electrochemical analyses coupled with MEC performance confirmed that anammox electrode biofilms were responsible for current generation through EET-dependent oxidation of NH4+. 15N-labelling experiments revealed the molecular mechanism of the EET-dependent anammox process. NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate when electrode was used as the terminal electron acceptor. Comparative transcriptomics analysis supported isotope labelling experiments and revealed an alternative pathway for NH4+ oxidation coupled to EET when electrode was used as electron acceptor. The results presented in my dissertation provide the first experimental evidence that marine and freshwater anammox bacteria can couple NH4+ oxidation with EET, which is a significant breakthrough that is promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen using bioelectrochemical systems.

碩士
逢甲大學
環境工程與科學學系
105
Anaerobic ammonium oxidation (Anammox) is an alternative nitrogen removal technology has paid close attention in recent years. Anammox is use of chemoautotroph bacteria in anaerobic environment with inorganic carbon as carbon source, and transform NH4-N and NO2-N into N2 and a little amount of NO3-N. The cost is lower than the traditional biological treatment. However, it has been found that the domestication of anaerobic ammonium oxidation bacteria is very difficult in recent years. That is a difficult problem to break through. The purpose of this study is to complete the domestication of anaerobic ammonium oxidation bacteria, using molecular biological detection technology as the core of the bacteria phase changes, and monitoring the water quality and observation of microbial changes during the experiment. Its desirable to detect flora by molecular biological detection technology. Demonstrate the presence of target bacteria, and to assess whether the objectives for the study were achieved. The results showed that short-term activation and rapid domestication of anaerobic ammonium oxidation bacteria is feasible. It had a clear trend in the four weeks. NH4-N and NO2-N are decrease in a short time and the ratio gradually approaching anaerobic ammonium oxidation reaction ratio 1.32. It proved that anaerobic ammonium oxidation is working. At the same time, it was observed that the red biofilm was exist in the reactor and pipe, which means that the anaerobic ammonium oxidation bacteria has been proliferated. However, the nitrogen load has also affected the reaction. If the nitrogen load increase, it may have the negative impact of the domestication process cause the NO3-N accumulating. Through the microscopic observation and molecular biology detection technology, we can know that the domesticated sludge is active and know the presence of anaerobic ammonium oxidation bacteria, which proves that the domestication is successful.

碩士
國立中興大學
環境工程學系所
97
The anaerobic ammonia oxidation (ANAMMOX) reaction is one of side pathways in the nitrogen cycle. The anaerobic ammonia oxidation bacteria could directly oxidized ammonium and nitrite to nitrogen gas under anoxic conditions by the following equation NH4+ + NO2- → N2 + 2H2O. Anammox reaction, which is different from the traditional nitrification and denitrification reaction, is important for the balance of the nitrogen cycle under lower oxygen concentration environment. In this research, the microbial community structures of different type anaerobic ammonia oxidation reactors(developed by ITRI) including upflow anaerobic sludge bed (UASB) system and partial nitrification/Anammox system with BioNET cotton that operated under various influent concentrations were studied using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)、Cloning and fluorescence in situ hybridization (FISH) techniques. Not only the relationship between the microbial community structures and nitrogen removal efficiency were compared, the possible roles of microorganisms co-existed with the anammox bacteria community were studied as well. Gene sequences obtained from DGGE patterns showed that anaerobic ammonia oxidation bacteria “Candidatus Brocadia fulgida” existed in the UASB system. Application of FISH quantification on both ammonia and nitrite oxidizing bacteria in the UASB indicated the existence of ammonia oxidizing β-proteobacteria(10%) and nitrite oxidizing bacteria Nitrobacter sp.(5%). We hypothesize that ammonia oxidizing bacteria converted ammonium to nitrite then being used by anammox bacteria to ensure total nitrogen removal. Results from DGGE experiments revealed that heterotrophic bacteria including Steroidobacter denitrificans、Clostridium celatum、Syntrophus sp.、Butyrivibrio fibrisolvens and Desulfobulbus sp. existed in the UASB system. We hypothesize that heterotrophic bacteria might be involved in the degradation of complex compounds in the UASB system to reduce the oxygen concentration and release carbon dioxide for aiding the survival of anammox bacteria. Ammonia oxidizing bacteria、nitrite oxidizing bacteria、Bacterium Ellin、Defluvicoccus vanus、Rubrivivax gelatinosus、Aquimonas sp. and certain uncultured bacterium existed in the Partial nitrification/Anammox with BioNET system. However, no anammox bacteria were detected. Candidatus Brocadia fulgida exhibited the common characteristics of anammox bacteria and its the biofilm aggregates could show strong autofluorescence. It is the only known anammox species exhibiting this feature. Detailed characteristics of this anammox bacterium remains unknown, so does research on the microbial community co-existed with Candidatus Brocadia fulgida. Further studies on Candidatus Brocadia fulgida as well as its interactions with the other microorganisms are needed.