No description available.

No description available.

No description available.

A detailed study has been carried out to demonstrate the importance of external and internal mass transfer on the nitrification rates in three distinct treatment processes: flocculent and granular activated sludge, and suspended carrier reactor (SCR) systems. The major emphasis was on external mass transfer, and the impact of system hydrodynamics on this mechanism. Laboratory-scale flocculent and granular sequencing batch reactors were operated for the nitrification of a synthetic wastewater. A two-stage, continuous, nitrifying SCR was operated using the same wastewater feed. Within each stage, biofilm was grown on two types of commercial carriers- the Natrix C10/10 from ANOX AB (Sweden); and the K1 carrier from Kaldnes Miljøteknologi (Norway). Biofilm carriers obtained from each of these reactors was utilised for the mass transfer investigations. The major findings, and contributions of the work to the field of biological wastewater treatment, are described in the following paragraphs. In order to complete the work, a novel experimental tool, the TOGA (Titrimetric and Off-Gas Analysis) sensor was created, which utilises off-gas mass balancing, coupled with pH titration to provide detailed measurement of biological reaction rates. An original method for off-gas mass balancing was developed, within a reactor that allowed modification of the hydrodynamic conditions using gas phase mixing independent of dissolved oxygen control within the liquid phase. This sensor has already proven to be a highly effective tool not only for the measurement of oxygen but also for carbon dioxide and various nitrogen species, and has application for numerous other compounds present in the gas phase of biological reactors (e.g. hydrogen, methane). The application of the TOGA sensor signals to the nitrification process was demonstrated, which enabled the online measurement of oxygen, ammonia, and nitrite reaction rates. The TOGA sensor development underpinned the majority of the subsequent experimental work within this thesis. Dissolved oxygen microelectrodes were also used, enabling microscale measurements to be made in conjunction with the macroscale TOGA sensor analyses. Combined with size and microbiological analyses a detailed study of mass transfer and reaction was able to be carried out on the various systems. For suspended aggregate systems (flocs and granules): A spherical particle model was developed and used to predict the potential for external mass transfer limitation in flocs and granules. The significance of this limitation was confirmed experimentally, by observing changes in reaction rate or concentration boundary layer (in the TOGA sensor or microelectrode study, respectively) upon modification of the system’s flow conditions. Despite this flow effect being small, and only observable under low bulk liquid substrate concentrations, the external mass transfer limitation was concluded to be significant for biological flocs and granules even at higher substrate concentrations. As particle size and the maximum volumetric reaction rate of the biomass increases, external mass transfer effects become increasingly significant. The work highlights the impact of mass transfer limitation on the measurement of Monod half saturation coefficients (KS) in flocs and granules. Without accounting for external or internal mass transfer limitation, KS is seriously overestimated and becomes a lumped parameter, reflecting not only the microbial response but also the mass transfer limitations observed within the system under study. To avoid confusion or generation of erroneous results, care should be taken in defining, measuring and utilising the half saturation coefficient in biological systems where the biomass is not present as individual cells or extremely small flocs. For Suspended Carrier Reactor systems: External and internal mass transfer are both concluded to be important rate limiting steps within suspended carrier reactors. The demonstration of a significant impact of fluid flow conditions on the nitrification rates highlights the impact of external mass transfer limitation within these systems. Application of a one-dimensional biofilm model to the experimental results led to the conclusion that there is little difference between the external mass transfer limitation of the two different carrier types, for carriers grown under the same environmental conditions. However, there was a significantly higher areal nitrification rate observed on the Natrix carriers compared to the Kaldnes carriers. It is the biofilm structure that is critically important in characterising the mass transfer steps. Systems operated under high nitrogen loads, producing filamentous biofilms on the carrier surface, were found to have larger external mass transfer coefficients and responses to changes in fluid flow than those carriers which were operated under nitrogen-limited conditions (producing a flatter, more gel-like biofilm). The structure of the biofilm colonising the carrier surface was far more important in defining the mass transfer coefficient than the actual carrier type used. In a remarkably similar trend to that of the external mass transfer coefficient, the biofilm morphology was again significantly more important than carrier type in determining both the magnitude and response to fluid flow of the gas-liquid mass transfer coefficient for oxygen (kLa) calculated within the laboratory TOGA sensor. These findings led to the postulation that direct gas-biofilm interfacial mass transfer mechanism is occurring within the SCR systems. This hypothesis is an alternative to the standard mechanism of gas transfer from the bubble into the liquid phase, and then into the biofilm. Understanding of interfacial transfer is likely to be important for developing the knowledge of SCR processes. Overall, both external and internal mass transfer phenomena have been demonstrated to create important rate limitations to suspended aggregate systems (flocs and granules) and biofilms grown in suspended carrier reactors. This significantly advances the conceptual understanding of these biological treatment processes.

A detailed study has been carried out to demonstrate the importance of external and internal mass transfer on the nitrification rates in three distinct treatment processes: flocculent and granular activated sludge, and suspended carrier reactor (SCR) systems. The major emphasis was on external mass transfer, and the impact of system hydrodynamics on this mechanism. Laboratory-scale flocculent and granular sequencing batch reactors were operated for the nitrification of a synthetic wastewater. A two-stage, continuous, nitrifying SCR was operated using the same wastewater feed. Within each stage, biofilm was grown on two types of commercial carriers- the Natrix C10/10 from ANOX AB (Sweden); and the K1 carrier from Kaldnes Miljøteknologi (Norway). Biofilm carriers obtained from each of these reactors was utilised for the mass transfer investigations. The major findings, and contributions of the work to the field of biological wastewater treatment, are described in the following paragraphs. In order to complete the work, a novel experimental tool, the TOGA (Titrimetric and Off-Gas Analysis) sensor was created, which utilises off-gas mass balancing, coupled with pH titration to provide detailed measurement of biological reaction rates. An original method for off-gas mass balancing was developed, within a reactor that allowed modification of the hydrodynamic conditions using gas phase mixing independent of dissolved oxygen control within the liquid phase. This sensor has already proven to be a highly effective tool not only for the measurement of oxygen but also for carbon dioxide and various nitrogen species, and has application for numerous other compounds present in the gas phase of biological reactors (e.g. hydrogen, methane). The application of the TOGA sensor signals to the nitrification process was demonstrated, which enabled the online measurement of oxygen, ammonia, and nitrite reaction rates. The TOGA sensor development underpinned the majority of the subsequent experimental work within this thesis. Dissolved oxygen microelectrodes were also used, enabling microscale measurements to be made in conjunction with the macroscale TOGA sensor analyses. Combined with size and microbiological analyses a detailed study of mass transfer and reaction was able to be carried out on the various systems. For suspended aggregate systems (flocs and granules): A spherical particle model was developed and used to predict the potential for external mass transfer limitation in flocs and granules. The significance of this limitation was confirmed experimentally, by observing changes in reaction rate or concentration boundary layer (in the TOGA sensor or microelectrode study, respectively) upon modification of the system’s flow conditions. Despite this flow effect being small, and only observable under low bulk liquid substrate concentrations, the external mass transfer limitation was concluded to be significant for biological flocs and granules even at higher substrate concentrations. As particle size and the maximum volumetric reaction rate of the biomass increases, external mass transfer effects become increasingly significant. The work highlights the impact of mass transfer limitation on the measurement of Monod half saturation coefficients (KS) in flocs and granules. Without accounting for external or internal mass transfer limitation, KS is seriously overestimated and becomes a lumped parameter, reflecting not only the microbial response but also the mass transfer limitations observed within the system under study. To avoid confusion or generation of erroneous results, care should be taken in defining, measuring and utilising the half saturation coefficient in biological systems where the biomass is not present as individual cells or extremely small flocs. For Suspended Carrier Reactor systems: External and internal mass transfer are both concluded to be important rate limiting steps within suspended carrier reactors. The demonstration of a significant impact of fluid flow conditions on the nitrification rates highlights the impact of external mass transfer limitation within these systems. Application of a one-dimensional biofilm model to the experimental results led to the conclusion that there is little difference between the external mass transfer limitation of the two different carrier types, for carriers grown under the same environmental conditions. However, there was a significantly higher areal nitrification rate observed on the Natrix carriers compared to the Kaldnes carriers. It is the biofilm structure that is critically important in characterising the mass transfer steps. Systems operated under high nitrogen loads, producing filamentous biofilms on the carrier surface, were found to have larger external mass transfer coefficients and responses to changes in fluid flow than those carriers which were operated under nitrogen-limited conditions (producing a flatter, more gel-like biofilm). The structure of the biofilm colonising the carrier surface was far more important in defining the mass transfer coefficient than the actual carrier type used. In a remarkably similar trend to that of the external mass transfer coefficient, the biofilm morphology was again significantly more important than carrier type in determining both the magnitude and response to fluid flow of the gas-liquid mass transfer coefficient for oxygen (kLa) calculated within the laboratory TOGA sensor. These findings led to the postulation that direct gas-biofilm interfacial mass transfer mechanism is occurring within the SCR systems. This hypothesis is an alternative to the standard mechanism of gas transfer from the bubble into the liquid phase, and then into the biofilm. Understanding of interfacial transfer is likely to be important for developing the knowledge of SCR processes. Overall, both external and internal mass transfer phenomena have been demonstrated to create important rate limitations to suspended aggregate systems (flocs and granules) and biofilms grown in suspended carrier reactors. This significantly advances the conceptual understanding of these biological treatment processes.

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.

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.