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.

The primary objective of this study was to evaluate the rates of nitrate removal for a nitrate containing, low organic carbon wastewater interacting with four different carbon-containing solid substrates (alder woodchips, corn silage, manure and woodchip biochar). Batch systems were tested for nitrate removal, and systems with a combination of three carbon substrates (75% woodchips, 12.5% silage, and 12.5% manure or woodchip biochar by mass) produced average nitrate removal rates of 571 and 275 mg-N L⁻¹ D⁻¹, and systems containing the carbon substrates individually produced rates between 11.4 - 3.3 mg-N L⁻¹ D⁻¹. Silage proved to be the dominant carbon substrate providing high quantities of organic carbon to fuel denitrification. With the introduction of semi-continuous flow, all systems had nitrate removal rates that converged to 13.3 – 6.4 mg-N L⁻¹ D⁻¹, which is approximately two orders of magnitude smaller than the rates of the mixture systems in the batch experiment. Silage appeared to be removed from of the systems with liquid exchange potentially causing the rate decreases. Columns filled with various volume fractions of woodchips (100%, 25%, 12.5%, and 0%) produced nitrate removal rates between 30.8 – 2.4 mg-N L⁻¹ D⁻¹ at a 24 hour and 12 hour hydraulic residence time (HRT). Greater nitrate removal was achieved with higher HRTs and larger fractions of woodchips (the 100% woodchip system at a 24 hour HRT produced the fastest nitrate removal rate of 30.8 mg-N L⁻¹ D⁻¹). When rates were normalized to the amount of woodchips in each column, higher efficiency was found in lower woodchip fraction systems (the 12.5% woodchip column produced the highest normalized nitrate removal rate of 56 mg-N L⁻¹ D⁻¹ L[subscript woodchips]⁻¹). Woodchips proved to be best suited as a long term carbon substrate for nitrate removal in a system containing a nitrate concentrated, low organic carbon wastewater. However, large amounts of woodchips were necessary to achieve nitrate removal greater than 50%. A 41 acre hypothetical wetland with a 3.3 day HRT and a nitrate influent concentration of 45 mg-N L⁻¹ would require 30,000 yd³ of woodchips to achieve 68% nitrate removal based on the values obtained in the bench scale column experiment. / Graduation date: 2012

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

Η μελέτη ενός πειράματος μικρής πιλοτικής κλίμακας, με δύο παράλληλα συστήματα ρηχών λεκανών (ύψους 0.35m), η μία λεκάνη με φύτευση του είδους Typha Latifolia και η άλλη χωρίς φύτευση, διεξάχθηκε για τον σχεδιασμό ελεύθερης επιφανειακής ροής (FWS) τεχνητού υγροτόπου. Οι δύο λεκάνες τροφοδοτήθηκαν με πραγματικά αστικά λύματα όπου οι χρόνοι παραμονής κυμάνθηκαν από 27,6 έως 38,0 ημέρες. Η μεταβολή του όγκου κάθε λεκάνης παρακολουθήθηκε για 2 συνεχή έτη και ταυτόχρονα υπολογίστηκαν οι ρυθμοί βροχόπτωσης και εξάτμισης. Η διαφορά του όγκου μεταξύ των δύο λεκανών οφειλόταν στην πρόσληψη νερού από τα φυτά, η οποία συγκρίθηκε με τις προβλέψεις της εξατμισοδιαπνοής παρόμοιων φυτών με την χρήση του υπολογιστικού προγράμματος REF-ET. Η συγκομιδή των φυτών πραγματοποιήθηκε τρείς φορές στην διάρκεια του 1ου έτους του πειράματος, ώστε να εκτιμηθεί ο ρυθμός πρόσληψης αζώτου από τα φυτά. Η σημαντικότερη διαφορά των δύο συστημάτων ήταν η αφαίρεση νερού μέσω της εξατμισοδιαπνοής των φυτών. Η πιλοτική μονάδα λειτούργησε έτσι ώστε να επιτευχθεί και απομάκρυνση της οργανικής ύλης (BOD5) και του ολικού αζώτου (TN) από τα λύματα. Ο σχεδιασμός της διευκόλυνε την ανάπτυξη ενός μαθηματικού μοντέλου, ακολουθώντας το πλαίσιο του μοντέλου της ενεργής ιλύος (ASM). Αρχικά το μαθηματικό μοντέλο αναπτύχθηκε για τις δύο λεκάνες με τις μικροβιακές διεργασίες που επικράτησαν στο εσωτερικό τους, ώστε να περιγραφεί πλήρως η συμπεριφορά τους. Η προσομοίωση και η εκτίμηση των παραμέτρων του μοντέλου επιτεύχθηκε με την χρήση του υπολογιστικού περιβάλλοντος του AQUASIM. Οι κύριες διεργασίες που ελήφθησαν υπόψη για την μοντελοποίηση ήταν η αμμωνιοποίηση, η αερόβια ετεροτροφική ανάπτυξη, η νιτροποίηση και η ανάπτυξη φυκών. Μια ισχυρή εποχική εξάρτηση παρατηρήθηκε για την συμπεριφορά κάθε λεκάνης όταν το μοντέλο εφαρμόστηκε για το 1ο έτος του πειράματος. Αυτό το μοντέλο επαληθεύτηκε ικανοποιητικά με τα πειραματικά δεδομένα του 2ου έτους. Η παρατηρούμενη μέση ετήσια απόδοση απομάκρυνσης του BOD5 και του TN ήταν 60% και 69%, αντίστοιχα για την λεκάνη χωρίς φυτά και 83% και 75%, αντίστοιχα για την λεκάνη με φυτά. Το μοντέλο προέβλεψε μέση ετήσια απόδοση απομάκρυνσης 82% για το BOD5 και 65% για το TN στην λεκάνη με φυτά, ικανοποιώντας τα κριτήρια για τον σχεδιασμό πλήρους κλίμακας τεχνητού υγροτόπου . Η ικανότητα του μοντέλου να προβλέπει όχι μόνο την απομάκρυνση της οργανικής ύλης αλλά και του ολικού αζώτου, θεωρήθηκε επαρκής όταν δοκιμάστηκε με έναν ελεύθερης επιφανειακής ροής τεχνητό υγρότοπο με 400 ισοδύναμο πληθυσμό, με μοναδική τροποποίηση τον συνυπολογισμό του περιορισμού του οξυγόνου στον ρυθμό της διεργασίας της νιτροποίησης. Επομένως, το δυναμικό μοντέλο διαμορφώθηκε με την ενσωμάτωση της πρόβλεψης του ρυθμού της εξατμισοδιαπνοής των φυτών και χρησιμοποιήθηκε για τον σχεδιασμό περίπτωσης μελέτης τεχνητού υγροτόπου πλήρους κλίμακας. Τα στοιχεία που απαιτούνται για αυτό τον σχεδιασμό περιλάμβαναν την παροχή εισόδου και κλιματολογικά στοιχεία (θερμοκρασίας και βροχόπτωσης) για την περιοχή του σχεδιασμού, καθώς και οι απαιτήσεις της ποιότητας εκροής. Η περίπτωση μελέτης για 4000 ισοδύναμο πληθυσμό όπου η ποιότητα εκροής ήταν σε μέσες ετήσιες τιμές BOD5=25mg/L και TN=15mg/L, χρειάστηκε μία συνολική επιφάνεια υγροτόπου 11 εκταρίων. Εάν χρησιμοποιηθούν δύο λεκάνες σε σειρά, η 1η με φυτά και η 2η χωρίς, τότε η συνολική επιφάνεια μειώνεται κατά περίπου 27%, ελέγχοντας μόνο την αρχική μέγιστη φύτευση της πρώτης λεκάνης του υγροτόπου. / The study at pilot-scale of two parallel systems with shallow basins (height h=0.35m), one planted with Typha Latiofolia and the other without vegetation, was conducted for the modeling of free water surface (FWS) constructed wetland systems. The basins were fed with real sewage at retention times ranging from 27.6 to 38.0 days. The variation of the volume in each basin was monitored for two consecutive years and simultaneously, rainfall and evaporation rates were calculated. The difference of the volume between the basins was due to the water absorption by the plants and was compared with the predictions of evapotranspiration rates of similar plants using the REF-ET calculation software. The harvesting of the plants was performed three times during the first year, in order to estimate the nitrogen uptake by the plants. The main difference in the two systems was the water removal through plant evapotranspiration. The pilot unit was operated so as to achieve the removal of both organic matter (BOD5) and total nitrogen (TN) from the sewage. Its design enabled the development of a mathematical model, following the framework of the activated sludge model (ASM). The simulation and the parameter estimation were achieved using the AQUASIM framework. The mathematical model describes the microbial processes, which dominated within the basins describing satisfactorily their behavior. The key processes accounted for in the modeling were ammonification, aerobic heterotrophic growth, nitrification and algal growth. A strong seasonal dependence was observed for each basin. The model was satisfactorily validated with the data of the second year. An observed average annual removal efficiency of BOD5 and TN were 60% and 69%, respectively for the basin without plants and 83% and 75%, respectively for the basin with plants. The model predicted average annual removal efficiency 82% for BOD5 and 65% for TN in the basin with plants, satisfying the design criteria of a full-scale constructed wetland. The ability of the model to predict not only the removal of organic matter but also total nitrogen removal, was considered sufficient as tested with a real free water surface constructed wetland of 400 population equivalent, with the sole modification being the inclusion of oxygen limitation in the nitrification rate. The dynamic model was amended with the direct incorporation of the plant evapotranspiration rate and it was used to design a full-scale constructed wetland. The required elements for this design included the inflow rate and climatic data (temperature and rainfall) for the design region, as well as the effluent quality requirements. In the case study of 4000 population equivalent, the effluent quality requirement was: average annual values for BOD5=25mg/L and for TN=15mg/L. The model was used to determine a total wetland surface requirement of 11ha. If two sequential basins are used, the first with plants and the second without, then the total wetland surface could be reduced by approximately 27%, controlling only the maximum initial vegetation in the first wetland basin.

Το Πι-κριτήριο των Bittanti et al. (1973) έχει χρησιμοποιηθεί εκτενώς σε εφαρμογές με στόχο την πρόβλεψη ενδεχόμενης βελτίωσης της απόδοσης ενός μη γραμμικού συστήματος υπό περιοδική είσοδο. Το κριτήριο όμως έχει τοπική ισχύ και περιορίζεται σε περιοδικές διαταραχές μικρού πλάτους. Η παρούσα εργασία αναπτύσσει μια μέθοδο προσδιορισμού διορθώσεων υψηλότερης τάξης στο πι-κριτήριο, προερχόμενη από βασικά αποτελέσματα της θεωρίας κεντρικής πολλαπλότητας (Center Manifold theory). Η προτεινόμενη μέθοδος βασίζεται στην επίλυση της μερικής διαφορικής εξίσωσης της κεντρικής πολλαπλότητας με χρήση δυναμοσειρών. Το τελικό αποτέλεσμα της προτεινόμενης προσέγγισης είναι ο κατά προσέγγιση υπολογισμός του δείκτη απόδοσης υπό μορφή σειράς, η οποία παρέχει ακριβή αποτελέσματα σε μεγαλύτερα εύρη. Η προτεινόμενη μέθοδος εφαρμόζεται σε έναν συνεχή αντιδραστήρα πλήρους ανάδευσης (CSTR), όπου στόχος είναι η μεγιστοποίηση της παραγωγής του επιθυμητού προϊόντος. Κατασκευάστηκε αλγόριθμος που προβλέπει την μόνιμη κατάσταση στην οποία καταλήγει ένα σύστημα απομάκρυνσης αζώτου που αποτελείται από αντιδραστήρα εμβολικής ροής και δεξαμενή δευτεροβάθμιας καθίζησης με ανακύκλωση. Με χρήση υπολογιστικού μοντέλου βασιζόμενο στο ASM3 υπολογίστηκαν οι μόνιμες καταστάσεις αυτού του συστήματος για ένα εύρος καταστάσεων λειτουργίας. Βρέθηκαν οι βέλτιστες τιμές των βαθμών ελευθερίας για την ελαχιστοποίηση του συνολικού αερισμού και για την ελαχιστοποίηση του συνολικού αζώτου στην απορροή. Και στις δύο περιπτώσεις στις βέλτιστες μόνιμες καταστάσεις παρατηρήθηκε έκπλυση των Nitrobacter δηλαδή παράκαμψη της παραγωγής των νιτρικών. / The frequency-dependent Pi criterion of Bittanti et al. (1973) has been used extensively in applications to predict potential performance improvement under periodic forcing in a nonlinear system. The criterion, however, is local in nature and is limited to periodic forcing functions of small magnitude. The present work develops a method to determine higher-order corrections to the pi criterion, derived from basic results of Center Manifold theory. The proposed method is based on solving the Center Manifold partial differential equation via power series. The end result of the proposed approach is the approximate calculation of the performance index in the form of a series expansion, which provides accurate results under larger amplitudes. The proposed method is applied to a continuous stirred tank reactor, where the yield of the desired product must be maximized. An algorithm was constructed, that predicts the steady state of a nitrogen removal system consisting of a plug flow reactor and a secondary clarifier with recycle. Using a numerical model based on ASM3 and a grid of degrees of freedom, the steady states of this system were calculated. The optimal values for minimizing the total aeration were found, as well as those for minimizing the total nitrogen exit flow. In both cases the Nitrobacter bacteria were washed out thus indicating the bypassing of nitrate production.

En aquesta tesis s'ha desenvolupat un sistema de control capaç d'optimitzar el funcionament dels Reactors Discontinus Seqüencials dins el camp de l'eliminació de matèria orgànica i nitrogen de les aigües residuals. El sistema de control permet ajustar en línia la durada de les etapes de reacció a partir de mesures directes o indirectes de sondes. En una primera etapa de la tesis s'ha estudiat la calibració de models matemàtics que permeten realitzar fàcilment provatures de diferents estratègies de control. A partir de l'anàlisis de dades històriques s'han plantejat diferents opcions per controlar l'SBR i les més convenients s'han provat mitjançant simulació. Després d'assegurar l'èxit de l'estratègia de control mitjançant simulacions s'ha implementat en una planta semi-industrial. Finalment es planteja l'estructura d'uns sistema supervisor encarregat de controlar el funcionament de l'SBR no només a nivell de fases sinó també a nivell cicle. / In this Thesis a control system has been developed which permits optimizing the performance of the Sequencing Batch Reactors (SBR) within the field of organic matter and nitrogen removal from the wastewater. This control system is based on the on-line adjustment of the length of the reaction phases using directly or indirectly the data acquired from the sensors. In a first stage of the Thesis the calibration of the activated sludge models is studied what permits obtaining models for testing different operating and control strategies. From the analysis of historical data several options for controlling the SBR are obtained and most suitable is tested using a simulation approach. Afterwards, the control strategy is implemented in a semi-industrial plant obtaining promising results. Finally, a proposal for a supervisory control system is presented which can be in charge of controlling the performance of the SBR not only at a phase level but also at cycle level.

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.

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.

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.

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.