The Chesapeake Bay watershed has seen an increase in population, nutrient loading, and stringent effluent limits; therefore, cost-effective technologies must be explored and implemented to intensify the treatment of regional wastewater. This work describes the bioaugmentation and retention of anammox (AMX) granules in a continuous adsorption/bio-oxidation (A/B) mainstream deammonification pilot-scale process treating domestic wastewater. The AMX granules were collected from the underflow of a sidestream DEMON® process. The bioaugmentation rate was based on several factors including full-scale sidestream DEMON® wasting rate and sidestream vs mainstream AMX activity. The retention of bioaugmented AMX granules required a novel settling column at the end of the deammonification step. The settling column was designed to provide a surface overflow rate (SOR) that allowed dense AMX granules to settle into the underflow and less dense floccular biomass to outselect into the overflow. B-Stage was operated to out-select nitrite oxidizing bacteria (NOB) by maintaining an ammonia residual (>2 mg NH4-N/L), a relatively high dissolved oxygen (DO) (>1.5 mg O2/L) concentration, an aggressive solids retention time (SRT) for NOB washout, and intermittent aeration for transient anoxia. AMX activity was not detected in the mainstream at any time. The settling column AMX retention quantification suggested but did not confirm AMX were maintained in the mainstream. NOB were not suppressed during this study and no nitrite accumulation was present in the mainstream process. It was theorized that AMX granules were successfully settled into the settling column underflow and accumulated in the intermittently mixed sidestream biological phosphorus reactor (SBPR) where they disintegrated. This work also describes optimization of carbon addition to an anoxic partial denitrification anammox (PdN/A) moving bed biofilm reactor (MBBR) testing glycerol, acetate, and methanol as carbon sources to maximize total inorganic nitrogen (TIN) removal through the anammox pathway and to minimize effluent TIN. A carbon feeding strategy was developed and was evaluated by the extent of partial denitrification vs full denitrification (partial denitrification efficiency, PdN efficiency). All three carbon sources were capable of high TIN removal, low effluent TIN, and moderate to high PdN efficiency. Average TIN removal for glycerol was 10.0 ± 3.6 mg TIN/L, for acetate it was 8.7 ± 2.9 mg TIN/L, and for methanol it was 11.5 ± 5.6 mg TIN/L. Average effluent TIN for glycerol was 6.0 ± 4.0 mg TIN/L, for acetate it was 5.0 ± 1.1 mg TIN/L, and for methanol it was 4.3 ± 1.5 mg TIN/L. Average PdN efficiency for glycerol was 91.0 ± 9.0%, for acetate it was 88.0 ± 7.7%, and for methanol it was 74.0 ± 8.5%. When PdN efficiency was factored into the cost of each carbon source, methanol was 5.83% cheaper than glycerol per mass TIN removed and 59.0% cheaper than acetate per mass TIN-N removed. / Master of Science / The Chesapeake Bay watershed has seen an increase in population, nutrient loading, and stringent effluent limits; therefore, cost-effective technologies must be explored and implemented to intensify the treatment of regional wastewater. This work involves removing nitrogen from wastewater in a pilot sized modeled from a real wastewater treatment plant. The removal of nitrogen from wastewater can become costly. This cost is due to aeration and chemical demands to remove the nitrogen. This masters work uses a type of microorganism that can remove nitrogen without the need for aeration or chemicals through anaerobic ammonia oxidation (AMX bacteria). A specific environment has been created for AMX bacteria during this study to ensure they perform nitrogen removal optimally. Often times, communities of bacteria can help remove nitrogen more effectively when they work together. Therefore, communities of bacteria were encouraged to grow during this study. We were able to see that nitrogen removal was indeed occurring at high rates and producing high effluent water quality. We used several different metrics to prove this nitrogen removal technology worked well. This research was important because it showed the capabilities of a highly intensified process of successful nitrogen removal at a pilot-scale facility. It is the hope that these findings can be improved upon and implemented at full-scale facilities. These full-scale facilities would be able to achieve low levels of nitrogen in their effluent while saving millions of dollars on operational costs.

Anaerobic ammonium oxidation (anammox) is a pathway that has been known for almost 2 decades, but few studies have investigated its importance in natural groundwaters. This thesis investigated the presence of anammox cells and the groundwater geochemistry of 2 sites (Elmira and Putnam) in southwestern Ontario where groundwaters are contaminated with high levels of nitrate and ammonium. Fluorescence in situ hybridization (FISH) was used to quantify the relative abundance of anammox cells in these waters. Our results showed that anammox cells could be detected in many wells at both sites and that their relative abundance varied between 0.45 and 4.81 % at the Putnam site, whereas it ranged between 0.8 to 8.4 % at the Elmira site. These values are within the same range as those obtained for marine and freshwater environments where anammox cells have been detected. In addition, indirect observations point to the fact that N cycling at the 2 sites might be linked to Fe and Mn reduction, but additional experiments are needed. In summary, our results corroborate the findings of N-labeled microcosm experiments which demonstrated that anammox was an important pathway of N cycling in those groundwaters and molecular analyses that detected important anammox organisms at the same sites.

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.

Anaerobic ammonium oxidation (anammox) is a pathway that has been known for almost 2 decades, but few studies have investigated its importance in natural groundwaters. This thesis investigated the presence of anammox cells and the groundwater geochemistry of 2 sites (Elmira and Putnam) in southwestern Ontario where groundwaters are contaminated with high levels of nitrate and ammonium. Fluorescence in situ hybridization (FISH) was used to quantify the relative abundance of anammox cells in these waters. Our results showed that anammox cells could be detected in many wells at both sites and that their relative abundance varied between 0.45 and 4.81 % at the Putnam site, whereas it ranged between 0.8 to 8.4 % at the Elmira site. These values are within the same range as those obtained for marine and freshwater environments where anammox cells have been detected. In addition, indirect observations point to the fact that N cycling at the 2 sites might be linked to Fe and Mn reduction, but additional experiments are needed. In summary, our results corroborate the findings of N-labeled microcosm experiments which demonstrated that anammox was an important pathway of N cycling in those groundwaters and molecular analyses that detected important anammox organisms at the same sites.

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.

Anaerobic ammonium oxidation (anammox) is a pathway that has been known for almost 2 decades, but few studies have investigated its importance in natural groundwaters. This thesis investigated the presence of anammox cells and the groundwater geochemistry of 2 sites (Elmira and Putnam) in southwestern Ontario where groundwaters are contaminated with high levels of nitrate and ammonium. Fluorescence in situ hybridization (FISH) was used to quantify the relative abundance of anammox cells in these waters. Our results showed that anammox cells could be detected in many wells at both sites and that their relative abundance varied between 0.45 and 4.81 % at the Putnam site, whereas it ranged between 0.8 to 8.4 % at the Elmira site. These values are within the same range as those obtained for marine and freshwater environments where anammox cells have been detected. In addition, indirect observations point to the fact that N cycling at the 2 sites might be linked to Fe and Mn reduction, but additional experiments are needed. In summary, our results corroborate the findings of N-labeled microcosm experiments which demonstrated that anammox was an important pathway of N cycling in those groundwaters and molecular analyses that detected important anammox organisms at the same sites.

The new Canadian federal wastewater regulations, which restricts the release of ammonia from treated wastewaters, has resulted in upgrade initiatives at many water resource recovery facilities across the country to reduce the discharge of ammonia into our natural waters. The objective of this dissertation is therefore to investigate and optimize the performance of two attached growth technologies for rural and peri-urban/urban municipal ammonia removal. In particular, the first specific objective of this dissertation is to investigate the performance and microbial response of the BioCord technology as an upgrade system for the post-carbon removal nitrification of rural wastewaters. The second specific objective is to study the start-up of an attached growth anammox technology to enhance current knowledge pertaining to anammox biofilm attachment, growth and maturation. The results pertaining to the first specific objective of this research, a study of the design and optimization of the BioCord technology, demonstrates a recommended design rate for the post-carbon removal, nitrifying BioCord system of a surface area loading rate (SALR) of 1.6 NH4⁺-N/m²·d and up to 1.8 NH4⁺-N/m²·d with steady ammonia-nitrogen removal efficiencies greater than 90% and steady and low solids production rate up to 0.26 g TSS/d. A loss of system stability and biofilm sloughing, identified as fluctuating ammonia removal rates and solids production rates, were observed at elevated SALRs of 2.1 and 2.4 g -N/m²·d. The microbial results indicate that the meso-scale structure of the biofilm and the micro-animal population are directly affected by operational conditions. Enhanced air scouring configuration is shown to be a potential optimization strategy to prevent the clogging of biofilm pores and improve the system stability in terms of solids production rate in the BioCord technology. The results pertaining to the second specific objective of this research, the study of inoculation and carrier modification strategies for the rapid start-up of attached growth anammox technology, demonstrates significantly higher kinetics, faster biofilm growth and greater anammox bacteria enrichment on the silica-functionalized carriers and pre-seeded denitrifying carriers in a system inoculated with detached anammox biofilm mass during the early stages of attachment and growth of start-up. The study suggests that the use of the silica-functionalized and pre-seeded denitrifying carriers along with detached anammox biofilm inoculation has the potential to accelerate the anammox biofilm attachment, growth and maturation.

Esta pesquisa objetivou enriquecer e purificar, em quimiostatos, consórcios microbianos capazes de oxidar amônia a nitrogênio (\'N IND.2\'), sob condições anaeróbias utilizando como inóculo: (Q1) biomassa de reator nitrificante-desnitrificante de estação de tratamento de água residuária de indústria produtora de aminoácidos (Ajinomoto) e (Q2) lodo granular de reator UASB de abatedouro de aves (Avícola DACAR, TIETÊ). Os inóculos foram enriquecidos em quimiostatos com vazão afluente de 18ml/h de meio basal específico com concentrações médias de 80 mg \'N-NH IND.4\'POT.+\'/L, 75,2 mg \'N-NO IND.2\'POT.-\'/L e 1000 mg/l de bicarbonato como única fonte de carbono. Nos quimiostatos Q1 e Q2 as eficiências médias de remoção foram de 51,6% e 39,3% de amônia e 60,5% e 53,2% de nitrito, respectivamente, após 296 dias de operação. A eficiência média de remoção de nitrogênio total (\'N-NH IND.4\'POT.+\' + \'N-NO IND.2\'POT.-\') foi de 57,1% e 43% após 296 dias de operação, respectivamente para Q1 e Q2. A purificação dos consórcios enriquecidos foi realizada utilizando-se metodologia de gradiente de densidade por centrifugação Percoll. Os consórcios microbianos foram observados por exames microscópicos e a diversidade foi avaliada por DGGE antes e depois da purificação pelo protocolo Percoll. O DGGE revelou mudança na estrutura dos consórcios presentes em Q1 e Q2 no decorrer do período de operação dos quimiostatos. A técnica de hibridação in situ (FISH) com sonda fluorescente (Amx - 368) confirmou a presença de microrganismos anammox nos dois consórcios microbianos. O seqüenciamento do DNA ribossomal 16S de bandas obtidas do gel de DGGE, utilizando-se primers universais para domínio Bacteria, relacionou por árvore de máxima verossimilhança, duas bandas com o grupo das bactérias verdes não-sulfurosas. / This research aimed to enrich and to purify, in chemostats, microbial trusts capable to realize ammonium oxidation to dinitrogen (\'N IND.2\') under anaerobic conditions, using as inoculum: (Q1) biomass from nitrifying-denitrifying reactor of wastewater treatment plant of amino-acids industry (Ajinomoto) and (Q2) granular sludge from upflow anaerobic sludge blanket UASB reactor treating poultry wastes - Avícola DACAR, TIETÊ. The inoculum was enriched in chemostats with affluent flow of 18 ml/h of specific basal media with mean concentrations of 80 mg \'N-NH IND.4\'POT.+\'/L, 75.2 mg \'NO IND.2\'POT.-\'/L and 1000 mg/l of bicarbonate as sole carbon source. In Q1 and Q2 chemostats the mean efficiency of removal were 51.6% and 39.3% of ammonium, and 60.5% and 53.2% of nitrite, respectively, after 296 days of operation. In Q1 and Q2 chemostats, the mean efficiency of total nitrogen removal (\'N-NH IND.4\'POT.+\' + \'NO IND.2\'POT.-\') were 57.1% and 43%, respectively, after 296 days of operation. The purification of enriched microbial trusts was carried following methodology of density gradient by centrifugation (Percoll). The microbial trusts were observed by microscopic analysis and the diversity was evaluated by DGGE, before and after the purification by the Percoll protocol. The DGGE analysis showed changes in microbial trusts structure in Q1 and Q2 in chemostats operation period. The fluorescence in situ hibridization technique (FISH) with Amx-368 probe confirmed the presence of anammox microrganisms in both microbial trusts. The sequencing of recovered bands of DGGE was carried through ribossomal DNA 16S using universal primers for bacteria Domain that related two bands with green nosulphur bacterium using maximum likelyhood tree.

Esta pesquisa objetivou enriquecer e purificar, em quimiostatos, consórcios microbianos capazes de oxidar amônia a nitrogênio (\'N IND.2\'), sob condições anaeróbias utilizando como inóculo: (Q1) biomassa de reator nitrificante-desnitrificante de estação de tratamento de água residuária de indústria produtora de aminoácidos (Ajinomoto) e (Q2) lodo granular de reator UASB de abatedouro de aves (Avícola DACAR, TIETÊ). Os inóculos foram enriquecidos em quimiostatos com vazão afluente de 18ml/h de meio basal específico com concentrações médias de 80 mg \'N-NH IND.4\'POT.+\'/L, 75,2 mg \'N-NO IND.2\'POT.-\'/L e 1000 mg/l de bicarbonato como única fonte de carbono. Nos quimiostatos Q1 e Q2 as eficiências médias de remoção foram de 51,6% e 39,3% de amônia e 60,5% e 53,2% de nitrito, respectivamente, após 296 dias de operação. A eficiência média de remoção de nitrogênio total (\'N-NH IND.4\'POT.+\' + \'N-NO IND.2\'POT.-\') foi de 57,1% e 43% após 296 dias de operação, respectivamente para Q1 e Q2. A purificação dos consórcios enriquecidos foi realizada utilizando-se metodologia de gradiente de densidade por centrifugação Percoll. Os consórcios microbianos foram observados por exames microscópicos e a diversidade foi avaliada por DGGE antes e depois da purificação pelo protocolo Percoll. O DGGE revelou mudança na estrutura dos consórcios presentes em Q1 e Q2 no decorrer do período de operação dos quimiostatos. A técnica de hibridação in situ (FISH) com sonda fluorescente (Amx - 368) confirmou a presença de microrganismos anammox nos dois consórcios microbianos. O seqüenciamento do DNA ribossomal 16S de bandas obtidas do gel de DGGE, utilizando-se primers universais para domínio Bacteria, relacionou por árvore de máxima verossimilhança, duas bandas com o grupo das bactérias verdes não-sulfurosas. / This research aimed to enrich and to purify, in chemostats, microbial trusts capable to realize ammonium oxidation to dinitrogen (\'N IND.2\') under anaerobic conditions, using as inoculum: (Q1) biomass from nitrifying-denitrifying reactor of wastewater treatment plant of amino-acids industry (Ajinomoto) and (Q2) granular sludge from upflow anaerobic sludge blanket UASB reactor treating poultry wastes - Avícola DACAR, TIETÊ. The inoculum was enriched in chemostats with affluent flow of 18 ml/h of specific basal media with mean concentrations of 80 mg \'N-NH IND.4\'POT.+\'/L, 75.2 mg \'NO IND.2\'POT.-\'/L and 1000 mg/l of bicarbonate as sole carbon source. In Q1 and Q2 chemostats the mean efficiency of removal were 51.6% and 39.3% of ammonium, and 60.5% and 53.2% of nitrite, respectively, after 296 days of operation. In Q1 and Q2 chemostats, the mean efficiency of total nitrogen removal (\'N-NH IND.4\'POT.+\' + \'NO IND.2\'POT.-\') were 57.1% and 43%, respectively, after 296 days of operation. The purification of enriched microbial trusts was carried following methodology of density gradient by centrifugation (Percoll). The microbial trusts were observed by microscopic analysis and the diversity was evaluated by DGGE, before and after the purification by the Percoll protocol. The DGGE analysis showed changes in microbial trusts structure in Q1 and Q2 in chemostats operation period. The fluorescence in situ hibridization technique (FISH) with Amx-368 probe confirmed the presence of anammox microrganisms in both microbial trusts. The sequencing of recovered bands of DGGE was carried through ribossomal DNA 16S using universal primers for bacteria Domain that related two bands with green nosulphur bacterium using maximum likelyhood tree.

Élimination de l'azote est obligatoire dans moderne usine de traitement des eaux usées (STEP) en raison de sa toxicité à la fois humaine et l'écosystème. Un niveau élevé d'azote peut provoquer une eutrophisation dans le système aquatique. Élimination de l'azote autotrophes qui combine nitritation partielle et Anammox est une technologie attrayante qui est approprié pour une grande force d'ammonium des eaux usées à faible teneur en carbone organique. Toutefois, le ralentissement de la croissance extrême des bactéries Anammox avec le temps de 9-13 jours doubler entrave la pleine application de l'échelle. L'objectif de cette recherche était d'étudier la faisabilité et la stratégie opérationnelle de l'enrichissement Anammox de boues conventionnelle aérobie (réacteur ASR), la dénitrification des boues (Réacteur DSR) et boue anaérobie (réacteur ANR) en utilisant réacteur biologique séquentiel (SBR). Anammox processus a été établi avec succès en DSR avec une élimination de l'azote total d'environ 80% sous le contrôle strict de l'oxygène au bout de 150 jours, ce qui est confirmé par la composition chimique de l'influent / effluents ainsi que l'analyse microbienne. Sous la même condition opérationnelle, ANR atteint seulement 20-30% d'élimination de l'azote total. Avec un temps plus court de rétention hydraulique (HRT) et de contrôle d'oxygène insuffisante, ASR a atteint 50-60% du total élimination de l'azote après 240 jours. Tous les réacteurs ont connu la fluctuation des performances au cours du processus d'enrichissement, qui est censé être la conséquence de facteurs inhibiteurs tels que l'oxygène dissous, sans nitrites et l'ammoniac libre ainsi que les bactéries coexistantes indésirables qui sont en concurrence pour le même substrat. L'électrophorèse sur gel de gradient dénaturant (DGGE) bande à partir des échantillons d'ADN amplifiés extraites ASR lors de l'étape d'enrichissement différente montre une nette évolution de la composition microbienne. Substances polymères extracellulaires (EPS) de différents biomasse Anammox ont été extraites et caractérisé par analyse quantitative et qualitative pour enquêter sur sa corrélation avec le processus d'enrichissement dans un bioréacteur de l'échelle du laboratoire. Une diminution de la protéine au polysaccharide (PN / PS) rapport et une augmentation du rendement total d'extraction EPS ont été observés au cours du processus d'enrichissement. La matrice à trois dimensions excitation d'émission (3D-EEM) a montré un endroit similaire des pics de fluorescence pour tous les échantillons tandis que les échantillons avec des bactéries Anammox possèdent deux pics distincts dans le bas de gamme d'onde d'excitation. Pics multiples excitation peuvent se produire comme en témoigne la fluorescence identique chromatogramme après chromatographie d'exclusion de taille (SEC) à séparation d'excitation / émission de 221/350 nm et 280/330 nm. Rayons ultraviolets (UV) absorbance à 210 nm a été enregistrée en même temps que la détection de fluorescence à une excitation / émission 222/300 nm de longueur d'onde, 221/350 nm et 280/330 nm après séparées par SEC. Avec l'enrichissement de bactéries Anammox, chromatogramme UV montré une augmentation en intensité et en nombre de pics, alors que chromatogrammes fluorescence ont montré nombre maximal similaire et seulement augmentent en intensité. Une augmentation de l'hydrophobie a été observée au cours du procédé d'enrichissement. Les résultats de cette étude sont attendus pour étendre la connaissance de l'évolution du processus d'enrichissement Anammox EPS ainsi que de fournir nouvelle approche pour la caractérisation des EPS extraites de Anammox boues / Removal of nitrogen is mandatory in modern wastewater treatment plant (WWTP) due to its toxicity to both human and the ecosystem. A high level of nitrogen may cause eutrophication in aquatic system. Autotrophic nitrogen removal which combines partial nitritation and Anammox is an attractive technology which is suitable for high Ammonium strength wastewater with low organic carbon content. However the extreme slow growth of the Anammox bacteria with doubling time of 9-13 days hinders its wide full scale application. The aim of this research was to investigate the feasibility and operational strategy of Anammox enrichment from conventional aerobic sludge (Reactor ASR), denitrification sludge (Reactor DSR) and anaerobic sludge (Reactor ANR) using sequencing batch reactor (SBR). Anammox process was successfully established in DSR with a total nitrogen removal of approximately 80% under strict oxygen control after 150 days which is confirmed by chemical composition of the influent/effluent as well as microbial analysis. Under the same operational condition, ANR reached only 20-30% total nitrogen removal. With a shorter hydraulic retention time (HRT) and insufficient oxygen control, ASR reached 50-60% total nitrogen removal after 240 days. All the reactors experienced fluctuating performances during the enrichment process, which is believed to be the consequence of inhibitory factors such as dissolved oxygen, free nitrite and free ammonia as well as undesirable coexisting bacteria which compete for the same substrate. The denaturing gradient gel electrophoresis (DGGE) band from the amplified DNA samples extracted from ASR during different enrichment stage shows a clear evolution of the microbial composition. Extracellular polymeric substances (EPS) from different Anammox biomass have been extracted and characterized by quantitative and qualitative analysis to investigate its correlation with the enrichment process in a lab scale bioreactor. A decrease of protein to polysaccharide (PN/PS) ratio and an increase in total EPS extraction yield were observed during the enrichment process. The three dimensional excitation emission matrix (3D-EEM) showed similar location of the fluorescence peaks for all samples while samples with Anammox bacteria possess two distinct peaks in the low excitation wavelength range. Multi-excitation peaks may occur as evidenced by the identical fluorescence chromatogram after size exclusion chromatography (SEC) separation at excitation/emission 221/350 nm and 280/330 nm. Ultraviolet (UV) absorbance at 210 nm was recorded simultaneously with fluorescence detection at excitation/emission wavelength 222/300 nm, 221/350 nm and 280/330 nm after separated by SEC. With the enrichment of Anammox bacteria, UV chromatogram showed increase in both intensity and number of peaks, whereas fluorescence chromatograms showed similar peak number and only increase in intensity. An increase of hydrophobicity was observed during the enrichment process. The results of this study are expected to extend the knowledge of EPS evolution of Anammox enrichment process as well as providing novel approach for the characterization of EPS extracted from Anammox sludge