The James River Treatment Plant (JRTP) operated a 2 MGD Integrated Fixed Film Activated Sludge (IFAS) demonstration process from November 2007 to April 2009 to explore IFAS performance and investigate IFAS technology as an option for a full scale plant upgrade in response to stricter nutrient discharge limits in the James River Basin. During the study, nitrification kinetics for both ammonia and nitrite oxidizing bacteria and plastic biofilm carrier biomass content were monitored on a near-weekly basis comparing the IFAS media, the IFAS process mixed liquor, and mixed liquor from the full-scale activated sludge process. Carrier biomass content is variable with respect to temperature and process SRT and relates to the localization of nitrification activity in the IFAS basin. Similar to trends observed for carrier biomass content (Regmi, 2008), ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) activity also shifted from the fixed film to the suspended phase as water temperatures increased and vice versa as the temperature decreased. The data suggest that AOB activity occurs on the surface of the biofilm carriers, while NOB activity remains deeper in the biofilm. During the highest temperatures observed in the IFAS tank, AOB activity on the media contributed as little as 30% of the total nitrification activity in the basin, and after temperatures dropped below 20 °C, AOB activity in the fixed film phase made up 75% of the total activity in the IFAS basin. During the warmest period of the summer, the media still retained more than 60% of the total NOB activity, and more than 90% of the total NOB activity during the period of coldest water temperature. This trend also points out that some AOB and NOB activity remained in the mixed liquor, even during the coldest periods. The retention of nitrification activity in the mixed liquor indicates that the constant sloughing of biomass off of the carriers allowed for autotrophic activity, even during washout conditions. Carrier biomass content and nitrification rates on the IFAS media remained constant along the length of the basin, indicating that the IFAS tank is will mixed with respect to biomass growth, although there was a concentration gradient for soluble species (NH₄-N, NO₂-N, NO₃-N). In addition to the weekly nitrification rate measurements, experiments were also conducted to determine how operational inputs such as dissolved oxygen (DO) and mixing affect the nitrification rates. Mixing intensity had a clear impact on nitrification rates by increasing the velocity gradient in the bulk liquid and decreasing the mass transfer boundary layer mass transfer resistance. At higher mixing intensities, advection through the mass transfer boundary layer increased making substrate more available to the biofilm. The affect of mixing was much more profound at low DO, whereas increased mixing had less effect on nitrification rates at higher bulk liquid DO. DO also affected nitrification rates, such that as DO increased it penetrated deeper into the biofilm increasing the nitrification rate in a linear fashion until the biofilm became saturated. Another aspect of the research was modeling effective half saturation effects for AOB and NOB activity in the fixed film phase. The modeling work demonstrated that KS for AOB activity on the media was similar to accepted suspended growth KS values, while KS for NOB activity on the media was considerably higher than suspended growth KS. This trend indicates that nitrite was not as bioavailable in the biofilm and resists diffusion into the deeper part of the biofilm where NOB activity takes place. KO for both AOB and NOB activity in the biofilm was higher than typical suspended growth values because of boundary layer and biofilm diffusion resistances. In addition, the presence of readily degradable organics did not significantly affect nitrification rates on the media, but did reduce nitrification rates in the mixed liquor. That, combined with low chemical oxygen demand (COD) uptake rates indicates that little heterotrophic activity is occurring on the media. / Master of Science

En aquesta tesi s’ha estudiat l’eliminació biològica de nitrogen en aigües residuals amb alta càrrega d’amoni. Aplicant certes condicions d’operació en un sistema de llots actius es pot aconseguir la nitrificació parcial o nitritació, que és l’oxidació de l’amoni a nitrit, evitant la conseqüent oxidació d’aquest nitrit a nitrat. Aquesta reducció en el procés de nitrificació aporta un seguit d’avantatges en front de la nitrificació convencional, tals com la reducció de les necessitats d’oxigen en la nitrificació i de la matèria orgànica en la desnitrificació, l’increment de la velocitat de desnitrificació i la reducció de la producció de biomassa. Aquesta tesi s’ha presentat com a compendi de publicacions. A continuació es presenta un breu resum dels articles: En l’Article I es presenta un sistema de nitrificació parcial format per una planta pilot amb tres reactors de fangs actius en sèrie. Aquesta planta pilot es va inocular a partir de llots d’una EDAR urbana i es va operar amb un llaç de control de la càrrega de nitrogen aplicada que consisteix en la modificació del cabal d’entrada segons els valors d’OUR mesurat als reactors. Utilitzant aquest llaç de control i treballant amb un temps de residència cel·lular baix es va aconseguir rentar els bacteris nitrit-oxidants del sistema. Simultàniament, també es va aconseguir tractar una elevada càrrega d’amoni i es va demostrar la viabilitat del procés a llarg termini tot obtenint una nitrificació parcial amb pràcticament només nitrit. Posteriorment, en l’Article II, es presenta la desnitrificació del corrent de nitrit obtingut amb el sistema desenvolupat en l’Article I. Aquesta desnitrificació es va realitzar utilitzant diferents fonts de carboni tals com etanol, glicerol, lixiviats d’abocador i finalment llots primaris i secundaris fermentats. Aquest estudi es va dur a terme en reactors discontinus seqüencials (SBR) i es van obtenir bones velocitats de desnitrificació per totes les fonts de carboni estudiades excepte pels llots secundaris fermentats. En l’Article III, es presenta l’estudi de l’efecte d’una llarga aturada del sistema de nitrificació parcial tot deixant d’alimentar durant 30 dies. Aquest estudi es va realitzar en quatre reactors discontinus que es van operar en diferents condicions d’aeració. Durant aquest període de temps es va fer un seguiment de l’activitat dels bacteris amoni-oxidants mitjançant respirometries i l’anàlisi FISH. Es va observar que és millor aturar el sistema de nitrificació parcial en condicions anòxiques i, a poder ser, no més de dues setmanes. Finalment, es va obtenir una recuperació ràpida del sistema utilitzant el llaç de control per OUR emprat en la planta pilot de l’Article I. Finalment, en l’Article IV es presenta l’estudi de l’efecte de les inhibicions per amoníac i per àcid nitrós en els bacteris amoni-oxidants, a més de l’efecte d’aquestes inhibicions en condicions de limitació per carboni inorgànic. Es va comprovar que la inhibició per amoníac es pot descriure amb precisió amb al model cinètic Haldane i la inhibició per àcid nitrós a un model d’inhibició no competitiva. Es va observar que l’efecte d’aquestes inhibicions s’incrementa en condicions de limitació per carboni inorgànic, sent molt més gran en el cas de l’àcid nitrós. / En esta tesis se estudia la eliminación biológica de nitrógeno en aguas residuales con una alta carga de amonio. Aplicando ciertas condiciones de operación en un sistema de lodos activos se puede conseguir la nitrificación parcial o nitritación, que es la oxidación del amonio a nitrito, evitando la consecuente oxidación de este nitrito a nitrato. Esta reducción en el proceso de nitrificación aporta unas ventajas frente a la nitrificación convencional, tales como la reducción de les necesidades de oxigeno en la nitrificación y de la materia orgánica en la desnitrificación, el incremento de la velocidad de desnitrificación y la reducción de la producción de biomasa. Esta tesis se presenta como compendio de publicaciones. A continuación se presenta un breve resumen de los artículos: En el Artículo I se presenta un sistema de nitrificación parcial formado por una planta piloto con tres reactores de lodos activos en serie. Esta planta piloto se inoculó a partir de lodos de una EDAR urbana y se operó con un lazo de control de la carga de nitrógeno aplicada que consiste en la modificación del caudal de entrada según los valores de OUR medidos en los reactores. Utilizando este lazo de control y trabajando con un tiempo de residencia celular bajo se consiguió lavar las bacterias nitritooxidantes del sistema. Simultáneamente, también se consiguió tratar una elevada carga de amonio y se demostró la viabilidad del proceso a largo plazo obteniendo una nitrificación parcial con prácticamente solo nitrito. Posteriormente, en el Artículo II, se presenta la desnitrificación de la corriente de nitrito obtenida con el sistema desarrollado en el Artículo I. Esta desnitrificación se realizó utilizando distintas fuentes de carbono tales como etanol, glicerol, lixiviados de vertedero y finalmente lodos primarios y secundarios fermentados. En este estudio se utilizaron reactores discontinuos secuenciales (SBR) y se obtuvieron buenas velocidades de desnitrificación para todas las fuentes de carbono estudiadas excepto para los lodos secundarios fermentados. En el Artículo III, se presenta el estudio del efecto de una larga parada del sistema de nitrificación parcial dejando de alimentar durante 30 días. Este estudio se realizó en cuatro reactores discontinuos que se operaron en distintas condiciones de aireación. Durante este período de tiempo se hizo un seguimiento de la actividad de las bacterias amonio-oxidantes utilizando técnicas respirométricas y el análisis FISH. Se observó que es mejor parar el sistema de nitrificación parcial en condiciones anóxicas y, a poder ser, no más de dos semanas. Finalmente, se consiguió una recuperación rápida del sistema utilizando el lazo de control por OUR utilizado en la planta piloto del Artículo I. Finalmente, en el Artículo IV se presenta el estudio del efecto de las inhibiciones por amoníaco y por ácido nitroso en las bacterias amonio-oxidantes, además del efecto de estas inhibiciones en condiciones de limitación por carbono inorgánico. Se comprobó que la inhibición por amoníaco se puede ajustar con precisión utilizando el modelo cinético Haldane y la inhibición por ácido nitroso utilizando el modelo de inhibición no competitiva. Se observó que el efecto de estas inhibiciones se incrementa en condiciones de limitación por carbono inorgánico, siendo mucho mayor en el caso del ácido nitroso. / Biological nitrogen removal of high-strength ammonium wastewater was studied in this thesis. Applying specific operational conditions in an activated sludge system, partial nitrification or nitritation (oxidation of ammonium to nitrite) was achieved, avoiding the consequently oxidation of this nitrite to nitrate. This reduction in the nitrification process provides some advantages in comparison to the complete nitrification process, such as the reduction of the oxygen requirements during nitrification and the organic matter during denitrification, the increasing of the denitrification rates and the reduction of the biomass production. This thesis was presented as a compendium of publications and a brief summary of the papers are presented: In Paper I, a partial nitrification system consisting of a pilot plant with three continuous stirred tank reactors in series is presented. This pilot plant was inoculated with sludge from a municipal WWTP and it was operated with a control loop of the nitrogen loading rate which was applied modifying the inflow rate depending on the OUR values measured in the reactors. With this control loop and low sludge retention time the washout of the nitrite-oxidizing bacteria was performed. Simultaneously, almost a complete partial nitrification to nitrite with a high nitrogen loading rate was achieved during a long term operation. Subsequently, in Paper II, the heterotrophic denitrification of the nitrite obtained with the pilot plant developed in Paper I was presented. Different organic carbon sources such as ethanol, glycerol, landfill leachate, fermented primary sludge centrate and fermented secondary sludge centrate were used in the heterotrophic denitrification. This study was carried out in sequential batch reactors (SBR) and a complete denitritation of a high-strength nitrite wastewater was achieved using these organic carbon sources with the exception of fermented secondary sludge centrate. In Paper III the study of the effect of a long-term starvation of a partial nitrification system during 30 days was presented. Four ammonium-starved reactors under different conditions of aeration were used. During this period the ammonia-oxidizing bacteria activity was evaluated using respirometric tests and the FISH analysis. It was observed that is better to shut-down a partial nitrification system under anoxic conditions and, if it is possible, no more than two weeks. Finally, a fast recovery of the system was achieved using the OUR control loop used in the pilot plant of Paper I. Finally, in Paper IV a study of the inhibitory effect by free ammonia and free nitrous acid on the ammonia-oxidizing bacteria under total inorganic carbon limitations and without total inorganic carbon limitations was presented. It was observed that the inhibition by free ammonia can be described accurately using the Haldane model and the inhibition by free nitrous acid using a non-competitive inhibition model. The effect of these inhibitions increased under total inorganic carbon limitation, being much higher in the case of the free nitrous acid.

I och med övergödningsproblematiken i Östersjön och Sveriges åtaganden i samband med Baltic Sea Action Plan kommer det i framtiden ställas högre krav på rening av kväve från de större avloppsreningsverken i Sverige. Anammox är processen där ammonium oxideras till kvävgas med nitrit som elektonacceptor. Anammox har under den senare tiden implementerats för rejektvattenbehandling i avloppsreningsverk runt om i världen.  När anammox används för att rena ammonium krävs att ca hälften av inkommande ammonium oxideras till nitrit genom nitritation. Detta steg utförs av ammoniakoxiderande bakterier (AOB) vilka kräver en aerob miljö. Den kombinerade processen med anammox och nitritation kallas deammonifikation. På Sjölunda avloppsreningsverk i Malmö har en pilotstudie, i sammarbete med VA-teknik på Lunds Tekniska Högskola, startats för att undersöka potentialen i att implementera deammonifikation i huvudströmmen på reningsverket. Detta examensarbete har syftat till att utarbeta metoder för aktivitetstest av anammox och AOB på bärarmaterial från pilotanläggningen. Med de utarbetade metoderna studerades sedan anammoxbakteriernas aktivitet vid förändrad temperatur. Eftersom anammox producerar kvävgas kunde en metod som baseras på tryckmätning utarbetas för att bestämma anammoxaktiviteten. Metoden visade sig vara tidseffektiv och enkel att utföra. I examensarbetet framkom resultat som visar på att anammox­aktiviteten är beroende av startkoncentrationen av nitrit vid startkoncentrationer under 75 milligram kväve per liter (mg N/l). Beroendet följer av diffusionsbegränsningar i biofilmen vid lägre koncentrationer. Vid startkoncentrationer i intervallet 75-150 mg N/l var aktiviteten oberoende av startkoncentrationen. Vid koncentrationer över 150 mg N/l konstaterades en aktivitetsminskning som troligtvis beror på nitritinhibering. Metoden som arbetades fram för aktivitetstest av AOB baserades på att mäta syrerespirationen innan och efter tillsats av ammonium i en alternerande syresatt reaktor. I metoden skulle en inhibitor för nitritoxiderande bakterier, NaClO3, tillsättas vid slutet av försöket för att säkerställa att dessa bakterier inte konsumerar syre och därmed påverkar resultatet. Vid tillsats av inhibitorn uppstod en orimligt stor aktivitetsförlust som tyder på att denna även inhiberar AOB.  Anammoxaktivitetens temperaturberoende analyserades genom aktivitetsmätning vid fyra temperaturer, i intervallet 11,1-24,9 °C. Försöken visade att förhållandet var exponentiellt och att vid en temperatursänkning från 24,9 °C till 11,1 °C förloras 93 % av aktiviteten. / Given the problem with eutrophication in the Baltic Sea and Sweden's obligations in the Baltic Sea Action Plan, a higher requirement on the removal of nitrogen from the major wastewater treatment plants is expected to be set in the future. Anammox is the process where ammonium is oxidized to nitrogen gas with nitrite as electron acceptor. Anammox has been implemented for treatment of the sludge liquor in wastewater treatment plants around the world. When anammox is used to reduce ammonium, the process requires about half the incoming ammonium to be oxidized into nitrite. This is conducted by ammonia oxidizing bacteria (AOB) that require an aerobic environment. The combined process with anammox and nitritation is called deammonification. A pilot study is taking place at Sjölunda Wastewater Treatment Plant in Malmö in collaboration with Water and Environmental Engineering, Department of Chemical Engineering, Lund University. The aim of the pilot study is to explore the potential of implementing deammonification in the main stream at the wastewater treatment plant. The aim of this Master Theisis is to develop methods for activity tests of Anammox and AOB on carrier material. The elaborated methods were then supposed to be used to study the change in activity with decreasing temperature.  Since anammox produces nitrogen gas a method based on pressure measurements was developed to determine the anammox activity. The results in this thesis showed that the anammox activity was dependent of the initial concentration of nitrite at concentrations below 75 mg N / L. This dependence is a result of the limitations of the diffusion in the biofilm at low concentrations. When the initial concentration of nitrite was within the range of 75 to 150 mg N / L the activity was independent of the initial concentration. At concentrations above 150 mg N / L there was a decrease in activity which probably occurred as a result of nitrite inhibition. To determine the activity of AOB a method based on the oxygen consumption rate was developed. The aeration switched between being turned on and off every five minutes and after some rounds of aereation, ammonium was added. The AOB activity was determined by calculating the difference between the oxygen consumption before and after the addition of ammonium. To ensure that nitrite-oxidizing bacteria (NOB) were not consuming any oxygen, NaClO3 was added at the end of the experiment. The resulting decrease in activity was too excessive to only represent the activity loss from only NOB which may suggest that NaClO3 also inhibits AOB. The temperature dependence of anammox activity was analyzed in activity tests at four temperatures in the range of 11.1 to 24.0°C. The experiments concluded that the relationship was exponential and by a drop in temperature from 24.9 ° C to 11.1 °C 93% of the activity was lost.

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.

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).

I Sverige finns det ungefär 25 000 riskklassade förorenade områden samt ännu fler områden som potentiellt är förorenade. Naturvårdsverket har tagit fram generella riktvärden för olika förorenade ämnen. Halter över riktvärdena antyder att risken för negativa effekter på människan eller miljön bedöms vara stor och att efterbehandling av området är aktuellt. Ett vanligt, men kostsamt efterbehandlingsalternativ är urschaktning och bortforsling av jord innehållandes halter av ämnen över riktvärdena. Ett intresse finns för att utveckla lätt tillämpbara metoder för platsspecifik riskbedömning av mark i syfte att skydda markmiljön och dess funktioner. Mikroorganismer som ingår i kvävets kretslopp utför nyckelfunktioner i jorden. Flera studier indikerar att dessa mikroorganismers abundans och aktivitet påverkas av flera olika markföroreningar. Inom forskningsprojektet APPLICERA har ett lysimeterexperiment genomförts där två jordar, med olika egenskaper med avseende på bland annat kornstorleksfördelning och pH (svagt basisk sandy loam respektive sur sand), förorenats med koppar respektive PAH:er i varierande halter. Halterna motsvarar riktvärden för känslig markanvändning (KM), mindre känslig markanvändning (MKM) och 3xMKM. Provtagning av jorden har skett vid tre tillfällen under en period av 16 månader. I detta examensarbete har nitrifierande mikroorganismers potential för att användas som indikatorer för förändrad markfunktion i förorenad jord undersökts. Detta har gjorts genom att kvantifiera ammoniakoxiderande arkéer (AOA), ammoniakoxiderande bakterier (AOB) samt de nitritoxiderande bakteriesläktena Nitrospira och Nitrobacter i jord som provtagits vid de olika tidpunkterna i lysimeterexperimentet. Mätning av potentiell ammoniakoxidation (PAO) har skett för jord från det sista provtagningstillfället. Resultaten visade att abundansen av mikroorganismerna generellt var större i den svagt basiska jorden klassificerad som sandy loam än i den sura jorden klassificerad som sand. Med tiden minskade AOA:s abundans för den högsta koncentrationen av koppar och PAH, särskilt utpräglad var minskningen i den sura jorden. Även Nitrospira minskade i abundans i PAH-förorenad jord för båda jordtyperna, dock kunde en minskning i abundans i kopparförorenad jord statistisk endast säkerställas för den sura jorden. AOB:s och Nitrobacters abundans visade inte på någon tydlig uppåt- eller nedåtgående trend varken över tid eller för ökande föroreningshalt. Skillnaden i kvantitet av mikroorganismer mellan jordtyperna antyder att jordens kemiska och fysiska egenskaper påverkade mikroorganismernas abundans såväl som föroreningarnas biotillgänglighet. PAO minskade med ökande kopparhalt för båda jordtyper, däremot sågs en minskning i PAO med ökande PAH-halt endast i den sura jorden. Resultaten antyder att abundansen av AOA och Nitrospiras verkar ha störst potential att utgöra generella indikatorer över ändrad markfunktion, dock lämpligen med komplettering av förbättrade mätningar av PAO för att få mer information om kvävets kretslopp i förorenad jord. / In Sweden there are around 25,000 contaminated areas classified as a risk for human health or the environment, and even more potentially contaminated areas. The Swedish Environmental Protection Agency has developed generic guideline values for hazardous substances.  Contamination levels exceeding the guideline values indicate a large risk for human health or the environment and that there is a need to remediate the site. A frequently used and expensive aftertreatment is excavation and removal of contaminated soil. Therefore, there is an interest to develop easy applicable methods for site-specific risk assessment with the purpose to protect the soil environment and its functions. Microorganisms involved in the nitrogen cycle perform key functions in soil. Several studies indicate that the abundance and activity of N-cycling microorganisms are sensitive to various soil pollution. Within the research project APPLICERA, a lysimeter experiment was conducted with two different soil types (acidic sandy soil and slightly alkaline sandy loam) and different contamination levels of copper and PAH. The contamination levels corresponded to generic guideline values for sensitive land-use (KM), less sensitive land-use (MKM) and 3xMKM. Soil samples were taken at three times during a period of 16 months. The aim of this master thesis was to investigate nitrifying microorganism potential as indicators for altered soil functioning in contaminated soil. Ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) and the nitrite-oxidizing bacteria genera Nitrospira and Nitrobacter has been quantified in soil samples with the different treatments. Measurements of the potential ammonia oxidation (PAO) have been performed in soil from the last sample taking.  The abundance of the nitrifying microorganisms was in general higher in the slightly alkaline sandy loam soil. Over time there was a decrease in AOA abundance for the highest concentration of copper and PAH, most pronounced in the acidic sandy soil. Also, a decrease in the abundance of Nitrospira in soil contaminated with PAHs could be seen for both soil types. In copper contaminated soil a distinct decrease in the abundance of Nitrospira could only be seen in the acidic sandy soil. The abundance of AOB and Nitrobacter did not show any obvious pattern for different contamination levels or over time. The difference in abundance of nitrifying microorganisms between the soil types suggests that the soil’s chemical and physical characteristics as well as the bioavailability of the contaminant affected their abundance. A significant decrease in PAO could be seen for the highest contamination level of copper for both soil types, but for the PAH contaminated soil PAO decreased only in the acidic sandy soil. These results suggest that the abundance of AOA and Nitrospira seems to have greatest potential as general indicators for altered soil functioning. However, improved activity measurements are needed to gain greater insight into the soil N-cycle functioning in contaminated soils.

Ammonium (NH4+) is a biological nutrient that is transformed in a wastewater treatment plant (WWTP) in a process called activated sludge. This is accomplished in an aerobic environment using microorganisms and inorganic carbon that convert the ammonium to nitrate (NO3-). This process is termed nitrification. Removal of ammonium is necessary due to its oxygen demand and toxicity to the environment. Nitrification is considered a slow process due to the slow growth rate of the nitrifying bacteria. Ammonia oxidizing bacteria (AOB) first covert the ammonium (NH4+) to nitrite (NO2-) followed by conversion to nitrate (NO3-) by nitrite oxidizing bacteria (NOB). These slow rates limit the treatment capacity of the WWTP. The initial hypothesis suggested that these slow rates were due to limited carbon in the aeration basin of a WWTP. A series of designed experiments and observational studies revealed substantial dissolved CO2 exists throughout a WWTP. Based on these findings, the central research focused on determining if an optimum dissolved CO2 concentration/ pH combination exists that maximizes nitrification. Experimentation conducted at a pH of 7.0 and varying concentrations of dissolved CO2 concentration revealed inhibition at low (<5 mg/l) and high (>30 mg/l) dissolved CO2 concentration levels. Further research found that optimum nitrification can be attained in a dissolved CO2 concentration range of 10 - 15 mg/l and a pH range of 7.5 - 8.0. A maximum specific growth rate of 1.05 - 1.15 days-1 was achieved. A partitioning of the sums of squares from these designed experiments found that pH accounts for approximately 83 percent of the sums of squares due to treatment with the dissolved CO2 concentration accounting for 17 percent. This suggests that pH is the dominant factor affecting nitrification when dissolved CO2 concentration is optimized. Analysis of the growth kinetics for two of the designed experiments was conducted. However, a set of parameters could not be found that described growth conditions for all operating conditions. Evaluating the results from these two experiments may suggest that a microbial population shift occurred between 16 and 19 mg/l of dissolved CO2 concentration. These dissolved CO2 concentrations represent pH values of 7.1 and 7.0, respectively, and were compared to experimentation conducted at a pH of 7.0. Though the pH difference is minor, in combination with the elevated dissolved CO2 concentration, a microbial shift was hypothesized. Microbial samples were collected from the designed experiment that optimized dissolved CO2 concentration (5, 10 and 15 mg/l) and pH (6.5, 7.0, 7.5 and 8.0). These samples were evaluated using Fluorescence in situ hybridizations (FISH) to determine the population density of common ammonium oxidizing bacteria (AOB) (Nitrosomonas and Nitrosospira) and nitrite oxidizing bacteria (NOB) Nitrobacter and Nitrospirae). The dominant AOB and NOB microbes were found to be Nitrosomonas and Nitrospirae. These results suggest that increased nitrification rates can be achieved by incorporating appropriate controls in a wastewater treatment plant (WWTP). With higher nitrification rates, lower nitrogen values can be obtained which will reduce the WWTP effluent nitrogen concentration. Conversely, these increased nitrification rates can also reduce the volume of an aeration basin given similar effluent nitrogen concentrations.

The accessory olfactory bulb (AOB) processes pheromonal signals which in turn drive social behaviors. Here we identify a tract of aromatase-expressing (arom+) fibers in the dorsal lateral olfactory tract (dLOT) which terminate in the granule cell layer (GCL) of the AOB. We utilized a retrograde tracer in aromatase reporter animals to delineate the source of these fibers. We show that these input fibers emerge almost exclusively from a contiguous population of arom+ neurons that spans the bed nucleus of the accessory olfactory tract (BAOT) and posterioventral subnucleus of the medial amygdala (MeApv). This population of neurons expresses the estrogen receptor alpha and contains more aromatase neurons in male mice than female mice. Thus, this population of feedback neurons can detect neuroendocrine changes and modulate the output of AOB projection neurons in a way that is sexually dimorphic and could influence every downstream target of the AOB.

No description available.

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.