Mine water effluent contains high levels of nitrogen due to residues from undetonated ammonium- nitrate based explosives. Excess nitrogen in aquatic ecosystems can cause eutrophication. Within a mining area, tailings and clarification ponds have the potential to reduce nitrogen levels by biological uptake of nitrogen into growing algae and denitrification in pond sediments. A previous study at the LKAB Kiruna mine investigated the potential nitrogen removal within the tailings and clarification ponds. The study showed that about 1-10 tonnes of nitrogen were removed each year, and that the removal by denitrification was limited by carbon.  The aim of this master thesis was to investigate if additions of different carbon compounds could improve the denitrification in sediment from the clarification pond at the LKAB Kiruna mine site. It was also of interest to see if the composition of the edogenous microbial community involved in nitrogen reduction changed after the treatments. Samples of sediment and pond water were collected in January 2014 and a laboratory experiment was set up where sediment and water was incubated with carbon additions under anoxic conditions. Three different carbon sources were tested: sodium acetate, hydroxyethyl cellulose and green algae. Pond water without additional carbon was used as a control. The sediment was incubated eight weeks at 20 °C with weekly water exchange and carbon addition. The removed water was analyzed to determine the amount of nitrogen removed. At start and after ending the incubation, potential denitrification in the sediment was determined with an enzymatic assay and the size of the genetic potential of nitrogen reduction was determined.  At start, the enzymatic assay showed that the potential denitrification rate in the sediment of the clarification pond at the LKAB Kiruna mine was not immediately enhanced by addition of carbon. However, during the incubation the removal of nitrate was enhanced by external carbon sources. Algae were a good carbon source, since the denitrifying community grew, the potential denitrification increased four times after incubation and the removal of nitrate was next to complete in the end of the incubation. The addition of cellulose also enhanced the denitrification activity to some extent and the abundance of genes coupled to denitrification increased. Further studies are needed to assess the practical use of external carbon sources like algae and plant material and how they would function in and potentially also affect a large, cold and complex system like the LKAB mining site.

Moving bed biofilm reactors (MBBRs) have been used effectively to reach low nutrient levels in northern Europe for nearly 20 years at cold temperatures. A relatively new technology to the US, the MBBR has most typically been used in a post-denitrification configuration with methanol for additional nitrate removal. Methanol has clearly been the most commonly used external carbon source for post-denitrification processes due to low cost and effectiveness. However, with the requirement for more US wastewater treatment plants to reach effluent total nitrogen levels approaching 3 mg/L, alternative electron donors could promote more rapid MBBR startup/acclimation times and increased cold weather denitrification rates. Bench-scale MBBRs evaluating four different electron donor sources, specifically methanol, ethanol, glycerol, and sulfide (added as Na2S), were operated continuously at 12 °C, and performance was monitored by weekly sampling and insitu batch substrate limiting profile testing. Ethanol and glycerol, though visually exhibited much higher biofilm carrier biomass content, performed better than methanol in terms of removal rate (0.9 and 1.0 versus 0.6 g N/m²/day.) Maximum denitrification rate measurements from profile testing suggested that ethanol and glycerol (2.2 and 1.9 g N/m²/day, respectively) exhibited rates that were four times that of methanol (0.49 g N/m²/day.) Sulfide also performed much better than any of the other three electron donors with maximum rates at 3.6 g N/m²/day and with yield (COD/NO₃-N) that was similar to or slightly less than that of methanol. Overall, the yield and carbon utilization rates were much lower than expected for all four electron donors and much lower than previously reported; indicating that there could be advantages for attached growth versus suspended growth processes in terms of carbon utilization rates. The batch limiting NO₃-N and COD profiles were also used to find effective K_s values. These kinetic parameters describe NO₃-N and COD limitations into the biofilm, which affect the overall denitrification rates. Compared to the other electron donors, the maximum rate for methanol was quite low, but the estimated K_s value was also low (0.4 mg/L N). This suggests high NO₃-N affinity and low mass transfer resistance. The other three electron donors estimated higher K_s values, indicating that these biofilms have high diffusion resistance. Biofilm process modeling is more complex than for mechanistic suspended growth, since mass transfer affects substrate to and into the biofilm. Simulating the bench-scale MBBR performance using BioWin 3.0, verified that μ_max and boundary layer thickness play key roles in determining rates of substrate utilization. Adjustments in these parameters made it possible to mimic the MBBRs, but it is difficult to determine whether the differences are due to the MBBR process or the model. / Master of Science

By rethinking wastewater treatment plants (WWTPs) as resource recovery facilities, it is possible to de- velop the next generation of WWTPs. Moreover, it allows to accomplish environmental goals, such as reducing the CO2 footprint, and comply with increasing effluent standards regarding the concentration of nitrogen in a more sustainable way. This research study aims to analyse the possibility of recirculating carbon within WWTPs in form of volatile fatty acids (VFAs) produced by co-fermentation of primary sludge and food waste. The obtained fermentation liquid is utilised as carbon source to enhance the denitrification process in a post-anoxic denitrification plant setup. Two pilot scale fermentation reactors were semi-continuously operated, systematically varying only in pH. By controlling one reactor to pH 10, while the second reactor was operated without pH control, it was possible to assess the influence of the pH on the carbon recovery process. Despite the pH not being controlled in the second fermentation reactor, it adjusted itself to a stable pH around 5.4. The co-fer- mentation process was monitored by weekly analysis of the SCOD and total amount of VFAs (TVFA). While the alkaline conditions in the reactor operated at pH 10 allowed a higher hydrolysis of the sub- strate, the second reactor, operated without pH control, achieved a more distinct acidification, due to the lower pH. Consequently, the SCOD in the reactor operated without pH control contains a higher percentage of TVFA amounting to 64 % in comparison to the reactor operated at pH 10 with 40 % TVFA. Furthermore, the achieved degree of fermentation was assessed by calculating the net increase of TVFA per gram of VS, respectively VSS. A higher degree of fermentation was achieved without pH control, resulting in a higher VFA yield compared to the fermentation reactor operated at pH 10. Moreover, anal- ysis of the individual VFAs by gas chromatography showed distinct differences in the composition of the fermentation liquids. According to the findings, the reactor operated at pH 10 produced mainly acetic acid (61 %), followed by propionic acid (18 %) and n-butyric acid (14 %). In contrast, the fermentation reactor operated without pH control produced mainly n-caproic acid (47 %), followed by acetic acid (25 %) and n-butyric acid (16 %). Despite the similar fermentation substrate supplied to both reactors, the acidic conditions in the reactor operated without pH control allowed carboxylic acid chain elongation from acetic acid to n-caproic acid, resulting in the main difference of the fermentation liquids. The fermentation liquid of the two reactors was filtered, diluted to a concentration of 5 g COD/L and supplied as additional carbon source to enhance denitrification in two continuously operated pilot scale moving bed biofilm reactors (MBBR), applying a carbon-to-nitrogen ratio of 4.5. One of the denitrifica- tion MBBRs received the carbon recovered by fermentation at pH 10 as external carbon source, whereby the carbon source produced by fermentation without pH control was supplied to the other MBBR. The maximal achieved denitrification rate was quite similar for both MBBRs amounting to 3.25 g NO3- Neq/(m2·d) for the MBBR receiving the carbon source recovered by co-fermentation at pH 10 and 3.38 g NO3-Neq/(m2·d) for the MBBR receiving the VFA-mix obtained by co-fermentation without pH control. However, the MBBR provided with the carbon source recovered by co-fermentation under acidic conditions achieved a higher average denitrification rate of 2.5 g NO3-Neq/(m2·d), compared with the MBBR receiving carbon produced by co-fermentation at pH 10 (1.8 g NO3-Neq/(m2·d)). The lower efficiency of the MBBR supplied with additional carbon recovered by fermentation at pH 10 is caused by an accumulation of NO2-N during the denitrification process. This accumulation of NO2-N indicates suboptimal conditions, both due to the composition of the supplied carbon source and an overall higher pH during the denitrification process, which might supress facultative anaerobes, such as denitrifiers. Nevertheless, this study shows that both VFA-rich carbon sources obtained by co-fermentation of pri- mary sludge and food waste are suitable to enhance denitrification of municipal wastewater, with the carbon source recovered by fermentation without pH control achieving a higher denitrification effi- ciency. / Eine Neuinterpretation kommunaler Klärwerke als Rohstoff-Rückgewinnungsanlagen ermöglicht die Entwicklung der Kläranlagen der Zukunft. Umweltziele, wie die Reduktion des CO2-Fußabdrucks und die Einhaltung steigender Abwasserstandards im Hinblick auf die Stickstoffkonzentration können somit nachhaltiger erreicht werden. Diese Forschungsstudie zielt darauf ab, die Möglichkeit der Rückführung von Kohlenstoff in Kläranlagen in Form leichtflüchtiger Fettsäuren (engl. volatile fatty acids, VFAs) zu untersuchen. Diese VFAs werden durch Co-Fermentation von Primärschlamm und Lebensmittelabfäl- len erzeugt und als zusätzliche Kohlenstoffquelle einer nachgeschalteten Denitrifikation zugeführt, um die Prozesseffizienz zu steigern. Zur Erzeugung der VFAs wurden zwei Fermentationsreaktoren halbkontinuierlich im Pilotmaßstab be- trieben, welche systematisch im pH-Wert variierten. Der Einfluss des pH-Wertes auf den Kohlenstoff- rückgewinnungsprozess konnte beurteilt werden, indem ein Reaktor auf pH 10 geregelt wurde, während dieser im zweiten Reaktor nicht beeinflusst wurde. In diesem stellte sich aufgrund ablaufender Reakti- onen ein stabiler pH-Wert um 5,4 ein. Der Co-Fermentationsprozess wurde durch wöchentliche Analyse des gelösten chemischen Sauerstoffbedarfs (engl. soluble chemical oxygen demand, SCOD) und der Ge- samtmenge an VFAs (TVFA) überwacht. Während die alkalischen Bedingungen in dem bei pH 10 be- triebenen Reaktor eine höhere Hydrolyse des Substrats ermöglichten, erreichte der zweite Reaktor auf- grund des niedrigeren pH-Werts eine stärkere Versäuerung. Folglich enthält der SCOD in dem Reaktor, der ohne pH-Regelung betrieben wurde, mit 64 % einen höheren Anteil an TVFA im Vergleich zu dem bei pH 10 betriebenen Reaktor mit 40 % TVFA. Außerdem wurde der erreichte Fermentationsgrad durch Berechnung der Nettozunahme der TVFA pro Gramm flüchtige Feststoffe (VS) bzw. flüchtige suspendierte Feststoffe (VSS) erfasst. Ein höherer Fer- mentationsgrad konnte ohne pH-Regelung erzielt werden, welche eine höhere VFA-Ausbeute im Ver- gleich zur Fermentation bei pH 10 zeigt. Deutliche Unterschiede in der Zusammensetzung der gewon- nenen VFAs konnten durch Analyse mittels Gaschromatographie erfasst werden. Demzufolge entstand bei der Fermentation bei pH 10 hauptsächlich Essigsäure (61 %), gefolgt von Propionsäure (18 %) und n-Buttersäure (14 %). Im Gegensatz dazu, produzierte der Fermentationsreaktor ohne pH-Regelung überwiegend n-Capronsäure (47 %), gefolgt von Essigsäure (25 %) und n-Buttersäure (16 %). Trotz des gleichen Fermentationssubstrates, welches beiden Reaktoren zugeführt wurde, ermöglichen die sauren Bedingungen in dem Fermentationsreaktor ohne pH-Regelung, eine Verlängerung der Carbonsäureket- ten von Essigsäure zu n-Capronsäure. Nach Filtration der in verschiedenen Milieus gewonnenen Fermentationssubstrate und Verdünnung auf eine Konzentration von 5 g COD/L, wurden diese zwei im Pilotmaßstab kontinuierlich betriebenen Fließbett-Biofilmreaktoren (engl. Moving bed biofilm reactor, MBBR) als zusätzliche Kohlenstoffquelle zur Denitrifikation zugeführt. Über die gesamte Versuchsdauer wurden ein MBBR mit dem alkalisch gewonnenen und der Andere mit dem im sauren Milieu erzeugten VFA-Mix betrieben. Das Kohlenstoff- Stickstoff-Verhältnis (C/N Ratio) lag dabei bei 4,5. Beide MBBRs wiesen eine vergleichbare maximale Denitrifikationsrate von 3,25 g NO3-Neq/(m2·d) (VFAs pH 10) und 3,38 g NO3-Neq/(m2·d) (VFAs pH un- geregelt) auf. Der MBBR, welcher die im sauren Milieu rückgewonnene Kohlenstoffquelle erhielt, er- reichte im Durchschnitt eine höhere Denitrifikationsrate von 2,5 g NO3-Neq/(m2·d) als der MBBR, der den bei pH 10 gewonnenen VFA-Mix erhielt (1,8 g NO3-Neq/(m2·d)). Die im Vergleich geringere Effizi- enz der alkalisch rückgewonnenen Kohlenstoffquelle wird durch eine NO2-N-Anreicherung während der Denitrifikation verursacht, welche suboptimale Bedingungen während des Prozesses indiziert. Dies ist sowohl auf die Zusammensetzung der zugeführten Kohlenstoffquelle, als auch auf einen insgesamt hö- heren pH-Wert während des Reduktionsprozesses zurückzuführen, der fakultative Anaerobier, wie bspw. Denitrifikanten, unterdrücken kann. Dessen ungeachtet zeigt diese Studie, dass beide durch Co- Fermentation von Primärschlamm und Lebensmittelabfällen gewonnenen VFA-reichen Kohlenstoff- quellen zur Verbesserung der Denitrifikation kommunalen Abwassers geeignet sind, wobei die durch Fermentation ohne pH-Regelung erzeugte Kohlenstoffquelle eine höhere Effizienz aufweist. / Det är möjligt att utveckla den nya generationen av avloppsreningsverk genom att ompröva avloppsreningsverk som resursanläggning. Därtill möjliggör det att uppnå miljömål som att minska koldioxidavtrycket och följa ökande utsläppskrav, t.ex. för kvävekoncentration, på ett mer hållbart sätt. Denna forskningsstudie syftar till att analysera möjligheten att återcirkulera kol inom reningsverket i form av lättflyktiga fettsyror (engl. volatile fatty acids, VFAs), producerades genom samfermentering av primärslam och matavfall. Det erhållna fermenteringssubstratet används som extern kolkälla för att förbättra processeffektiviteten i en efterdenitrifikationsanläggning. Två pilotskaliga fermenteringsreaktorer drevs i semikontinuerligt driftläge med endast en skillnad i pH. Det var möjligt att utvärdera pH-påverkan på kolåtervinningsprocessen genom att kontrollera pH- värdet i en reaktor till pH 10, medan den andra reaktorn drevs utan pH-kontroll. På grund av reaktionerna som fortlöpte, justerade sig den icke-kontrollerade reaktorn själv till ett stabilt pH runt 5,4. Samfermenteringsprocessen övervakades genom veckoanalys av kemisk syreförbrukning i filtrerade prover (engl. soluble chemical oxygen demand, SCOD) och total mängd av VFAs (TVFA). Medan den alkaliska miljö i den första reaktorn gynnade en högre hydrolys av substratet, uppnådde den andra reaktorn en mer tydlig surgöring på grund av det lägre pH-värdet. Följaktligen innehåller SCOD i reaktorn som drivs utan pH-kontroll en större andel TVFA – 64 % av SCOD - jämfört med reaktorn som drivs vid pH 10, där TVFA utgör 40 % av SCOD. Vidare analyserades den uppnådda fermenteringsgraden genom att beräkna nettoökningen av TVFA per gram VS, respektive VSS. En högre jäsningsgrad uppnåddes i sur miljö, vilket resulterade i en högre VFA-produktion jämfört med fermenteringsreaktorn som drevs vid pH 10. Därtill visade analys med gaskromtografi av de individuella VFA tydliga skillnader i sammansättning av substraten. Enligt rönen producerade reaktorn vid pH 10 mestadels ättiksyra (61 %) följt av propionsyra (18 %) och n-smörsyra (14 %). Däremot producerade fermenteringsreaktorn utan pH-kontroll mestadels n-kapronsyra (47 %) följt av ättiksyra (25 %) och n-smörsyra (16 %). Detta visar att trots att samma fermentationssubstrat användes för båda reaktorerna möjliggör den sura miljön i reaktorn utan pH-kontroll karboxylkedjeförlängningen från ättiksyra till n-kapronsyra. Fermentationssubstraten av de två reaktorerna filtrerades, utspäddes till en koncentration av 5 g COD/L och tillfördes som extern kolkälla, med ett kol/kväve-förhållande på 4,5, för att förbättra denitrifikationen i två kontinuerliga drivna biofilmreaktorer med rörliga bärare (engl. moving bed biofilm reactor, MBBR). En MBBR erhöll under hela experimentets gång den kolkälla som bildats under alkaliska förhållanden och den andra MBBR:en erhöll motsvarande kolkälla som bildats i den sura miljön i fermenteringsreaktorn utan pH-kontroll. Den maximala uppnådda denitrifikationskapaciteten var ganska likartad för båda MBBR: 3,25 g NO3-Neq/(m2·d) för den MBBR som opererades med den alkaliska erhållen kolkällan och 3,38 g NO3-Neq/(m2·d) för MBBR som erhöll den utspädda fermenteringsvätskan bildad utan pH-kontroll. Emellertid uppnådde den MBBR som erhöll kolkällan bildad i sura miljön en högre genomsnittlig denitrifikationskapacitet på 2.5 g NO3-Neq/(m2·d) jämfört med MBBR som fick kolkällan producerad genom fermentering vid pH 10 (1.8 g NO3-Neq/(m2·d)). Den lägre effektiviteten i den MBBR som fick den alkaliskt erhållna kolkällan orsakas av en ansamling av NO2-N under denitrifikationsprocessen, vilket indikerar suboptimala förhållanden. Detta beror både på sammansättningen av den tillförda kolkällan och ett högre totalt pH-värde under reduktionsprocessen, vilket kan hämma fakultativa anaerober såsom denitrifierare. Trots det visar denna forskningsstudie att båda de VFA-rika kolkällorna erhållna genom samfermentering av primärslam och matavfall är lämpliga för att förbättra denitrifikationen av kommunalt avloppsvatten, varvid kolkällan som produceras genom fermentering utan pH-kontroll uppnår en högre denitrifikationseffektivitet.

More stringent requirements on nitrogen removal from wastewater are the motivation for this thesis. In order to improve treatment results and enhance cost-efficient operation of wastewater treatment plants, model based control strategies are presented. A Java based simulator for activated sludge processes (JASS) is presented. The graphical user interface, educational experiences and implemented control strategies are discussed. Controlling the addition of an external carbon source is the next topic discussed. A simple model based feedforward controller is derived and evaluated in a simulation study. The controller attenuates process disturbances quickly. Further, two feedforward controllers for adjusting the aeration volume in activated sludge processes are derived. The aim of the volume control strategies was to efficiently dampen the impact of process disturbances without using an excessively high dissolved oxygen concentration. The simulation results are promising and show that the aeration volume may be a feasible control variable. A linearisation method for static input non-linearities is presented. The method gives essentially the same result as the existing standard method, but possesses some implementational advantages. The method is used to linearise the non-linear oxygen transfer function of an activated sludge process in an application study. Multivariable interactions in a process model describing nitrate removal in an activated sludge process are studied using the well known RGA method as well as a relatively novel tool based on Hankel norms. The results of the analysis are compared to conclusions drawn from common process knowledge and are used to design a multivariable control strategy. It was found that process disturbances may be rejected faster using multivariable control. Finally, the operational costs of the denitrification process are investigated and visualised graphically. Cost optimal regions are found by a numerical grid search. Procedures for controlling the denitrification process in a cost-efficient way are described.

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<p>More stringent requirements on nitrogen removal from wastewater are the motivation for this thesis. In order to improve treatment results and enhance cost-efficient operation of wastewater treatment plants, model based control strategies are presented.</p><p>A Java based simulator for activated sludge processes (JASS) is presented. The graphical user interface, educational experiences and implemented control strategies are discussed.</p><p>Controlling the addition of an external carbon source is the next topic discussed. A simple model based feedforward controller is derived and evaluated in a simulation study. The controller attenuates process disturbances quickly. Further, two feedforward controllers for adjusting the aeration volume in activated sludge processes are derived. The aim of the volume control strategies was to efficiently dampen the impact of process disturbances without using an excessively high dissolved oxygen concentration. The simulation results are promising and show that the aeration volume may be a feasible control variable.</p><p>A linearisation method for static input non-linearities is presented. The method gives essentially the same result as the existing standard method, but possesses some implementational advantages. The method is used to linearise the non-linear oxygen transfer function of an activated sludge process in an application study.</p><p>Multivariable interactions in a process model describing nitrate removal in an activated sludge process are studied using the well known RGA method as well as a relatively novel tool based on Hankel norms. The results of the analysis are compared to conclusions drawn from common process knowledge and are used to design a multivariable control strategy. It was found that process disturbances may be rejected faster using multivariable control.</p><p>Finally, the operational costs of the denitrification process are investigated and visualised graphically. Cost optimal regions are found by a numerical grid search. Procedures for controlling the denitrification process in a cost-efficient way are described.</p>

The gap between resource demand for the industrialized world and non-renewable feedstock like fossil fuels, essential agricultural fertilizer is getting increasingly severe, which has resulted in alarming-increasingly impacts on worldwide environmental problems. Meanwhile, wastewater treatment plants (WWTPs), as an essential facility for urbanization, is also confronting new rising challenges such as energy consumption and operation costs rather than only improving effluent wastewater quality. It is thereby important to develop new approaches for next-generation WWTPs with less energy consumption and costs in a sustainable way. The objective was to study the application of volatile fatty acids (VFAs) from waste as an external carbon source on denitrification by manometric tracking method in the lab-scale. Food waste and primary sludge were used for anaerobic fermentation to produce VFAs, which was then used as a sole external carbon source with various C/N ratio in denitrification batch test. The results were compared with traditional external carbon sources, acetate and methanol. It was found that the maximum denitrification rate with VFAs as an external carbon source was 15.73 ± 0.95 mg NOx-N/g VSS h, faster than those with acetate and methanol as external carbon sources. When C/N ratio ≥ 4.5 nitrate removal efficiency and the highest maximum denitrification rate were reached, the optimum C/N ratio for dosing VFAs as an external carbon source was thus determined as 4.5. In addition, denitrification capacity experiments with addition of VFAs produced from three different pH-controlled digestion reactors as an external carbon source were then conducted under an optimum C/N ratio of 4.5. As a result of the composition difference between VFAs produced from different pH environment, it was observed that, with VFAs from pH-10 digestion reactor denitrification rate was slightly higher than those with VFAs from pH-5 and none-pH-controlled digestion reactors. Furthermore, denitrification batch test using chemical tracking method was conducted to compare with manometric tracking method, and it was proven that the results obtained from manometric denitrification tests were reliable and valid. The overall results show that VFAs produced from anaerobic fermentation are an excellent external carbon source for denitrification, and it realizes the utilization of carbon resource recovery from WWTPs, which is crucial for next-generation wastewater treatment. / Klyftan mellan resursefterfrågan i den industrialiserade världen och icke-förnybar råvara som fossila bränslen, essentiell jordbruksgödsel, blir allt svårare, vilket har resulterat i oroväckande effekter på globala miljöproblem. Samtidigt står avloppsreningsverk (ARV), som är en viktig anläggning för urbanisering, inför nya ökande utmaningar som energiförbrukning och driftkostnader snarare än att förbättra kvaliteten på renat avloppsvatten. Det är därmed viktigt att utveckla nya strategier för nästa generations ARV med mindre energiförbrukning och kostnader på ett hållbart sätt. Målet var att studera tillämpningen av flyktiga fettsyror (VFA) från avfall som en extern kolkälla i denitrifikation med manometrisk metod i laboratorieskala. Matavfall och primärslam användes för anaerob rötning för att producera VFA, som sedan användes som en extern kolkälla med olika C/N-förhållanden i denitrifikationstest. Resultaten jämfördes med traditionella externa kolkällor, acetat och metanol. Det visade sig att maximal denitrifikationskapacitet med VFA som extern kolkälla var 15,73 ± 0,95 mg NOx-N/g VSS h, snabbare än de med acetat och metanol som extern kolkälla. När C/N-förhållandet ≥4,5 uppnåddes högsta nitratborttagningseffektivitet och maximala denitrifikationskapacitet, varför det optimala C/N-förhållandet för dosering av VFA som extern kolkälla bestäms som 4,5. Dessutom genomfördes experiment med denitrifikationskapacitets med tillsats av VFA producerat från tre olika pH-kontrollerade rötningsreaktorer som externa kolkällor under ett optimalt C/N-förhållande av 4,5. Som ett resultat av kompositionsskillnaden mellan VFA producerade från olika pH-miljöer, det observerades att med VFA från pH-10 kontrollerad reaktor denitrifikationskapacitet var något högre än med VFA från pH-5 och en icke-pH-kontrollerad reaktor. Vidare utfördes denitrifikationstest med användning av kemisk metod för jämförelse med manometrisk metod, och det visades att resultat erhållna från manometrisk denitrifikationstest var tillförlitliga och giltiga. De övergripande resultaten visar att VFA som produceras från anaerob rötning är en utmärkt extern kolkälla för denitrifikationsprocessen, vilket betyder att utnyttjandet av kolresursåtervinning från AVR är avgörande för nästa generations avloppsrening.

The aim of this thesis was to identify the denitrifying microbial communities in activated sludge from full-scale treatment plants and from small-scale reactors exposed to acetate or methanol as external carbon sources. Biological denitrification is currently the most widely used, sustainable and cost-effective process to remove nitrogen from wastewater. Increasingly strict effluent discharge standards are posing significant challenges to plant operators to reduce effluent NO3--N concentrations to levels as low as 2-3 mg L-1 or even lower. The lack of sufficient influent carbon in many municipal wastewater treatment plants makes it very difficult to achieve such low NO3--N concentrations in the effluent. An effective solution to the problem is to introduce additional external carbon sources to enhance denitrification. The selection of external carbon sources is not purely based on costs but is also dependent on the possible microbial transformations that these carbon sources may bring about in activated sludge. The most common carbon source used is methanol due to its low cost, but it has been found to cause long delays until an improvement in denitrification performance is observed. On the other hand, acetate has been found to improve denitrification almost instantaneously when added, but it has a significantly higher cost. In this study, methanol and acetate utilising denitrifiers were investigated in activated sludge with and without enrichment in laboratory scale bioreactors. The relevant denitrifiers were identified and evaluated in situ using culture independent methods particularly stable isotope probing (SIP), 16S rDNA cloning, fluorescence in situ hybridisation (FISH) and microautoradiography (MAR). Activated sludge collected from a biological nutrient removal plant exhibiting good denitrification was enriched in an anoxically-operated sequencing batch reactor (SBR) by feeding methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was operated over a duration of 7 months and the SBR denitrification rate improved from 0.02 mg NO3--N mg mixed liquor volatile suspended solids (MLVSS)-1 h-1 to a steady-state value of 0.06 mg NO3-N mg MLVSS-1 h-1. At steady state operation the enriched biomass was subjected to SIP with 13C-methanol to biomark the denitrifiers capable of utilising methanol under anoxic conditions. The separated 12C-DNA and 13C-DNA fractions from the SIP experiment were individually subjected to full cycle rRNA analysis. The dominant 16S rRNA gene phylotype (Group-A clones) in the 13C-library was closely related to the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96-97% sequence identities), while the most abundant clone groups in the 12C-library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes were designed for FISH to target the Group-A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes on SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, no correlation was found between denitrification rate and the relative abundances of the well known denitrifying genera Hyphomicrobium and Paracoccus nor the Saprospiraceae-clones visualised by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [14C] methanol uptake in the enriched biomass. As observed in full-scale operations, the methanol-fed SBR experienced a lag period of several weeks before denitrification performance increased. Using FISH quantification, it was shown that this coincided with the lag phase in the growth of the DEN67-targeted denitrifying population. It was therefore concluded that the Methylophilales bacteria dominant in our SBR system are likely to be important in full-scale methanol-fed denitrifying sludges. The acetate utilising microbial consortium in activated sludge was investigated without prior enrichment using stable isotope probing (SIP). 13C-acetate was used in SIP to biomark the DNA of the denitrifiers. The extracted 13C-DNA fraction was subjected to a full cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C-library were closely related to bacterial families Comamonadaceae and Rhodocyclaceae of class Betaproteobacteria (96-97% sequence identities). Seven oligonucleotide probes (DEN444, DEN220, DEN581, DEN441, DEN124, DEN220a and DEN1454) for use in FISH was designed to specifically target the identified phylotypes. Application of these probes on the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated over a duration of 16 days indicated a strong correlation between the level of CFDSBR denitrification and relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH combined with microautoradiography (FISH-MAR) further confirmed that the DEN581- and DEN124-targeted cells dominating the CFDSBR were capable of taking up [14C] acetate under anoxic conditions. The initial occurrence of the DEN444- and DEN1454-targeted bacteria and the final dominance of DEN581- and DEN124-targeted bacteria in the CFDSBR community were likely related to the changing in-reactor nitrite concentrations during the first few days of CFDSBR operation. Hence, the DEN444- and DEN1454-targeted bacteria were hypothesised to have low affinities for nitrite while DEN124- and DEN581-targeted bacteria have higher nitrite affinities. However, it was clear that all probe-targeted bacteria were denitrifiers capable of utilising acetate as a carbon source. The rapid increase in numbers of the probe-targeted organisms positively correlates with the immediate increase in denitrification rates. The rapid response and community shifts observed when acetate was used to enhance denitrification suggest that an intermittent application of acetate is quite effective to temporarily enhance the denitrification capacity of a treatment plant. However, the importance of a bacterial impact assessment of activated sludge subjected to intermittent acetate supplementation is recommended prior to the wide use of acetate in the wastewater industry. The acetate utilising denitrifying microbial communities investigated in the previous chapter were characterised according to their eco-physiological properties using the r- and K-selection criteria. The electron donor (acetate) and acceptor (nitrite) affinities of these probe-identified denitrifiers were used as traits for this characterisation. The substrate to microorganism (S/M) ratio was manipulated to provide high and low substrate concentrations in the reactor to create conditions favourable for r- and K-strategists, respectively. Two factors, namely feeding regimes and sludge retention times, were studied to achieve the desired S/M ratios and enable r/K characterisation. The high substrate affinities and high specific growth rates of two probe-identified denitrifiers (DEN124 and DEN581) did not enable resolution of these two organisms with the feeding regimes used in this study. However, the application of different sludge retention times as a control strategy to maintain constant high and low in-reactor S/M ratios enabled characterisation of the two probe-targeted denitrifiers DEN124 and DEN581 as K- and r-strategists, respectively. The in-reactor S/M ratios applied in this study did not facilitate the characterisation of populations targeted by probes DEN444 and DEN1454. The minor fluctuations of the S/M ratios during a cycle in the SBR operation was considered as a drawback, but conclusive results could still be obtained from the study. A chemostat reactor operation with constant loading and variable flow rates is suggested as an alternative. Conclusively, this study was able to identify specific groups of denitrifying microorganisms in activated sludge when exposed to acetate and methanol. Unlike most previous studies, which relied on culture dependent methods, this study adopted a pure culture independent approach to identify microorganisms in relation to their function, i.e. denitrification. Moreover, acetate denitrifiers were in situ characterised based on eco-physiological properties. The identification of denitrifying communities in this study has paved the way to a larger research project on the optimisation of denitrification processes with external acetate, methanol and other carbon supplements. As such, this study has contributed significantly to the understanding of the denitrification processes by linking process data with microbial investigations.