The success of enhanced biological phosphorus removal depends on the constant availability of volatile fatty acids (VFAs). To reduce costs of purchasing external carbon, waste streams would be a preferred source for nutrient removal. VFAs were shown to vary in the incoming sewage and fermentate from primary sludge (PS). Another available source of organic to generate VFAs is waste activated sludge (WAS). The effect of solids retention time and biomass concentration, as well as the effect of temperature and requirement for mixing on generation of VFA from the fermentation of WAS were investigated. It was found that VFA yields from sludge fermentation increased with SRT. At the longest SRT of 10 days improved biomass degradation resulted in the highest soluble to total COD ratio and the highest VFA yield. WAS fermentation was found highly temperature-dependent. The overall VFA–COD concentration in the non-mixed reactors was much lower than the mixed reactors. The study of fermentation of PS, WAS and a mixture of WAS and PS demonstrated that PS fermentation predictably generated a significantly higher amount of soluble COD than WAS. Co-fermentation of WAS with PS enhanced soluble COD production and increased the release of phosphate and ammonium. Fermentation of combined PS and WAS sludge generated a concentration of phosphate high enough to allow phosphorus recovery as struvite The effect of using glycerol as an external carbon source in biological phosphorus removal was investigated. Using glycerol directly resulted in the failure of the process which maintained enhanced biological phosphorus removal. When glycerol was co-fermented with waste activated sludge, significant VFA production was observed. By 2 | P a g e supplying the system with the VFA-enriched supernatant of the fermentate, biological phosphorus removal was enhanced. It was concluded that, if glycerol was to be used as external carbon source for biological phosphorous removal, the effective approach was to ferment glycerol with waste activated sludge. According to the cost analysis, the economic benefit of WAS fermentation can be demonstrated in three ways: 1) cost saving in external carbon addition; 2) cost saving in sludge handling; 3) revenue from phosphorus. At current condition, the value of the recovered P product is insignificant relative to the cost of chemicals that required for recovery and capital cost of the facilities. However, P recovery becomes important when the sustainability take into account.

The success of enhanced biological phosphorus removal depends on the constant availability of volatile fatty acids (VFAs). To reduce costs of purchasing external carbon, waste streams would be a preferred source for nutrient removal. VFAs were shown to vary in the incoming sewage and fermentate from primary sludge (PS). Another available source of organic to generate VFAs is waste activated sludge (WAS). The effect of solids retention time and biomass concentration, as well as the effect of temperature and requirement for mixing on generation of VFA from the fermentation of WAS were investigated. It was found that VFA yields from sludge fermentation increased with SRT. At the longest SRT of 10 days improved biomass degradation resulted in the highest soluble to total COD ratio and the highest VFA yield. WAS fermentation was found highly temperature-dependent. The overall VFA–COD concentration in the non-mixed reactors was much lower than the mixed reactors. The study of fermentation of PS, WAS and a mixture of WAS and PS demonstrated that PS fermentation predictably generated a significantly higher amount of soluble COD than WAS. Co-fermentation of WAS with PS enhanced soluble COD production and increased the release of phosphate and ammonium. Fermentation of combined PS and WAS sludge generated a concentration of phosphate high enough to allow phosphorus recovery as struvite The effect of using glycerol as an external carbon source in biological phosphorus removal was investigated. Using glycerol directly resulted in the failure of the process which maintained enhanced biological phosphorus removal. When glycerol was co-fermented with waste activated sludge, significant VFA production was observed. By 2 | P a g e supplying the system with the VFA-enriched supernatant of the fermentate, biological phosphorus removal was enhanced. It was concluded that, if glycerol was to be used as external carbon source for biological phosphorous removal, the effective approach was to ferment glycerol with waste activated sludge. According to the cost analysis, the economic benefit of WAS fermentation can be demonstrated in three ways: 1) cost saving in external carbon addition; 2) cost saving in sludge handling; 3) revenue from phosphorus. At current condition, the value of the recovered P product is insignificant relative to the cost of chemicals that required for recovery and capital cost of the facilities. However, P recovery becomes important when the sustainability take into account.

This project utilizes hydrocyclones in wastewater treatment to select for heavier solids, and has been used before in multiple small-scale systems. This is the first implementation of hydrocyclones in a full-scale plant for the purpose of increased settleability, while also achieving enhanced biological phosphorus removal without the use of an anaerobic selector. Hydrocyclones receive mixed liquor tangentially and separate light solids from more dense solids through their tapered shape, increasing the velocity of liquid as it moves downward and allowing for selection of a certain solids fraction. The hydrocyclones receive flow from the waste stream, selecting for dense solids to recycle through the process while light solids are wasted, creating a balance of granules and flocs with superior settling characteristics in which phosphorus is removed through phosphorus accumulating organisms (PAO). This project was implemented at a wastewater treatment plant rated at 20 MGD utilizing a 4-stage Bardenpho configuration with an IFAS system. This plant routinely experienced moderate settleability issues with an average SVI of 141 and a 90th percentile SVI of 179. Over time data was collected to characterize settleability and activity of PAO, GAO, and filaments. Using an external selector to achieve biological phosphorus is significant in that most wastewater treatment plants cannot do this without the use of an anaerobic selector. This has the potential to apply external selectors to existing infrastructure throughout plants worldwide to achieve not only biological phosphorus removal, but also improved settleability with a very minor capital investment. / Master of Science

Enhanced Biological Phosphorus Removal (EBPR) is an established process in wastewater treatment that uses bacteria to reduce phosphorus levels below regulatory discharge limits. Recently, in light of growing political concern over phosphorus sustainability, EBPR has also been recognised as a platform from which phosphorus may be recovered and recycled onto land as fertiliser. Operating EBPR to optimise performance and efficiency is therefore extremely important, but remains a challenge due to poor understanding of these bacterial ecosystems. Raman spectroscopy is a non-invasive, label-free, culture-independent technique capable of analysing live, single cells. Despite its advantages, Raman spectroscopy has been applied to study EBPR bacteria in just a handful of studies and thus has a low profile in this field of research. More work is required to investigate potential areas of application for Raman spectroscopy in EBPR research. The principal thesis presented here is that Raman spectroscopy could be used as a tool to improve EBPR. The Raman spectra used for this investigation were acquired from individual EBPR bacteria dried onto a calcium fluoride substrate. The bacterial samples were collected from three different sources, namely lab-scale sequencing batch reactors located in Edinburgh (University of Edinburgh, UK) and Boston (Northeastern University, USA), and a full-scale EBPR plant in Slough (Thames Water, UK). Using these spectra, some potential applications and limitations of Raman spectroscopy for improving EBPR were explored. In this foundation work, a particular emphasis on spectral analysis methods was kept in light of the benefits of automating analysis as well as the need for standardisation to be able to compare results between different studies and groups. Nine methods were compared for baselining Raman spectra of individual EBPR bacteria. From these, the “small-window moving average” (SWiMA) method was determined to be the best baselining technique for our purposes at the current time. In agreement with earlier studies, the Raman spectroscopic signatures of three key EBPR metabolites – polyphosphate, polyhydroxyalkanoate (PHA) and glycogen – were shown to be clearly identifiable in individual EBPR bacteria when present. The Raman shifts of characteristic spectral bands arising from polyphosphate were shown to vary significantly between samples and the implications of this were discussed. Examples of how the Raman spectra of individual bacteria can be modelled with multivariate tools to open up new areas for research were given. MCR modelling was demonstrated to offer a novel way to normalise the Raman spectra of individual EBPR bacteria prior to quantitative analysis. With the instrumental set-up in this work, the limit of detection (LOD) of aqueous polyphosphate samples was estimated to be approximately 0.08 M and 0.02 M for 10 second and 200 second acquisitions respectively. Future work is required to research ways in which a more comparable form of polyphosphate ‘standard’ might be prepared so that direct correlation can be drawn between measurements made on such a standard and measurements made in bacterial cells. Overall, several applications and challenges of Raman spectroscopy for the investigation of EBPR bacteria are presented in this work together with recommendation for how to process the spectral data. The conclusions drawn from this work indicate that Raman spectroscopy could be used as a tool to improve EBPR but further work is required to refine and apply these methods.

Low dissolved oxygen (DO) biological nutrient removal (BNR) is becoming a viable option to improve the energy efficiency of BNR. To properly model and design BNR processes for low DO operation, it is critical to fully understand how nitrifier, denitrifier, and polyphosphate accumulating organism (PAO) oxygen kinetics adapt in a shift from traditional DO operation (2 mg O2/L or more) to low DO operation. Research characterizing how oxygen kinetics shift over time in activated sludge biomass adapting to low DO is limited. Therefore, a method to characterize oxygen kinetics for nitrifiers, denitrifiers, and PAOs simultaneously is lacking. Here a method was developed to simultaneously measure the oxygen kinetics of nitrifiers, denitrifiers, and PAOs. This method, termed the SND and P-Uptake Oxygen Kinetics test, was able to estimate the ammonia oxidizing bacteria (AOB) oxygen half-saturation coefficient, ammonia maximum removal rate, denitrifier oxygen inhibition coefficient, total inorganic nitrogen (TIN) maximum removal rate, PAO oxygen half-saturation coefficient, phosphorus maximum uptake rate, and a simultaneous nitrification and denitrification (SND) optimum operation point. Three tests were conducted on the Virginia Initiative Plant (VIP) BNR Activated Sludge Pilot while it was operating at a process DO of 2 mg O2/L, and one test while it was operating at 1.5 mg O2/L. The measurements among the three initial tests showed high similarity in their parameter estimates. Estimated oxygen half-saturation and oxygen inhibition coefficients were compared to current suggested ranges and were within the expected magnitudes. At 2 mg O2/L, denitrifier oxygen inhibition coefficients and PAO oxygen half-saturation coefficients were estimated to be remarkably low here, under 0.4 and 0.1 mg O2/L, respectively. AOB oxygen half-saturation coefficients were variable here in the range of 0.62 to 2.57 mg O2/L, seeming to vary with available ammonia concentrations. Upon comparison with a previously developed respirometric test for nitrifier oxygen kinetics, termed the Declining DO test, the AOB oxygen half-saturation coefficient from the SND and P-Uptake Oxygen Kinetics test and the Declining DO test, when both were conducted on the VIP BNR Pilot, showed a similar trend. This provided validation for the AOB oxygen kinetics here and the usefulness of the test developed here. Additionally, measuring and plotting AOB and denitrifier oxygen kinetics together produced an intersection point where ammonia removal rates were equal to TIN removal rates. This intersection point was an optimum point for SND during the conditions of the test. This method can be used to characterize and track oxygen kinetic changes in a BNR system adapting from high to low DO. / Master of Science / Aerating biological processes in wastewater treatment plants is necessary to facilitate nitrogen and phosphorus removal but is extremely costly. Traditional dissolved oxygen concentrations in these processes are 2 mg O2/L or higher. Operating processes with low dissolved oxygen (DO) concentrations, less than 1 mg O2/L, can cut costs significantly. However, designing processes at low DO concentrations requires knowledge of how microorganisms utilize substrate with lower oxygen availability and how substrate utilization develops when gradually decreasing the DO concentration in a process. Here, a method was developed to measure the parameters describing the relationship between substrate utilization and DO concentration for the microorganisms responsible for nitrogen removal (nitrifiers and denitrifiers) and phosphorous removal (polyphosphate accumulating organisms). Additionally, the method provides an optimum DO setpoint for simultaneous nitrification and denitrification (SND) during testing conditions. This method, termed the SND and P-Uptake Oxygen Kinetics test, was able to estimate the following parameters simultaneously: ammonia oxidizing bacteria (AOB) oxygen half-saturation coefficient, ammonia maximum removal rate, denitrifier oxygen inhibition coefficient, total inorganic nitrogen (TIN) maximum removal rate, PAO oxygen half-saturation coefficient, and phosphorus maximum removal rate. Three tests were conducted on the Virginia Initiative Plant (VIP) BNR Activated Sludge Pilot while it was operating at a process DO of 2 mg O2/L, and one test while it was operating at 1.5 mg O2/L. The measurements among the three initial tests showed high similarity in their parameter estimates. Estimated oxygen half-saturation and oxygen inhibitions coefficients were compared to current suggested ranges and were within the expected magnitudes. Upon comparison with a previously developed test for nitrifier oxygen kinetics, termed the Declining DO test, the AOB oxygen half-saturation coefficient from the SND and P-Uptake Oxygen Kinetics test and the Declining DO test when both were conducted on the VIP BNR Pilot showed a similar trend, providing validation for the usefulness of the test developed here.

The goal of this research was to characterize the effects of temperature changes on the denitrification process in a biological nutrient removal (BNR) system. Specifically, there were three objectives. First, the effects of temperature changes on denitrification rates by a bacterial population from a BNR system were investigated. Next, the role which PHAs (poly-beta-hydroxyalkanoates) played in the denitrification process were examined. Finally, the effect of temperature changes on the production and consumption rates of PHAs was determined. Sacrificial batch experiments were performed to assess the kinetic and chemical trends present in the denitrification process. Mixed liquor from the last anaerobic zone of a pilot scale BNR system was injected into vials. These vials were pre-purged with nitrogen gas in order to prevent dimolecular oxygen (02) from being entrained in the mixed liquor. Next, the vials were placed on a shaker table for 30 minutes in order to allow all external COD to be consumed. Following this, each vial was injected with nitrates and various macronutrients. This process was repeated for three different sets of batch tests; each set was identical except for the added substrate. One set received no added substrate while the other two received either acetate or glucose. Vials were sacrificed over a period of three hours and analyzed for nitrate, phosphate, PHB (polybeta-hydroxybutyrate), PHV (poly-beta-hydroxyvalerate), glucose and acetate. / Master of Science

Toxic shock events can be detrimental to wastewater treatment systems and can result in long-term losses of system performance. If warned of an impending toxic shock, operators would have the opportunity to implement process controls that could help mitigate the effects of the shock event. The objective of this project was to evaluate the effectiveness of a developed corrective action strategy (involving aerobic endogenous respiration) on an enhanced biological phosphorus removal (EBPR) wastewater treatment plant (WWTP) shocked with chlorine. Three identical, laboratory-scale systems were designed to mimic one train of the Long Creek Water Resources Reclamation Facility (WRRF, Gastonia, NC). The basis of this study is a comparative performance analysis among the three trains; a negative control (unshocked and operated normally), a positive control (shocked with hypochlorite and operated normally), and the corrective action (shocked with hypochlorite and process controls implemented). Comparative performance analysis among the three trains was based on effluent quality, performance stability, and biomass kinetics as indicated by rates of respiration and phosphate release and uptake. The shock event and corrective action strategy both inhibited EBPR. After an initial perturbation, the positive control matched the performance of the negative control. The corrective action, however, exhibited significant instability in EBPR performance. Regardless of whether aerobic or anaerobic sludge storage conditions are selected, endogenous respiration will still result in system instability. It is recommended, therefore, that measures be taken to avoid imposing endogenous conditions on isolated sludge during a short-term toxic shock event. / Master of Science

Grundämnet fosfor är essentiellt för alla levande organismer men kan i överskott leda till problem med övergödning. Det finns därför höga krav på halten avloppsreningsverk släpper ut till recipienter. Idag sker stora delar av fosforreningen kemiskt genom dosering av fällningskemikalier. Det finns dock fördelar med att istället använda en biologisk metod som bygger på att reningsförhållandena premierar tillväxt av bakterier med möjlighet att ta upp mer fosfor än de behöver för sin cellväxt.  Bakterierna gynnas genom omväxlande anaeroba och aeroba zoner samt en god tillgång på lättillgänglig kolkälla och fosfor. Många reningsverk kombinerar den kemiska och biologiska fosforreningen men de är inte alltid kompatibla och den kemiska kan störa ut den biologiska. På Kungsängsverket finns sedan 2010 förutsättningar för en biologisk fosforreduktion men processen har inte fungerat tillfredsställande. Anledningen tros vara höga halter järn i slammet. Järnet fäller delar av den fosfor som är nödvändig för processen. Arbetet har därför syftat till att undersöka om det går att tvätta bioslammet på järn och på så sätt nå en fungerande fosforrening; vid vilka järnhalter detta sker och vilka besparingar det skulle kunna leda till för Uppsala Vatten och Avfall AB. För vidare utredning genomfördes ett pilotförsök där två reaktorer byggdes, en referensreaktor och en försöksreaktor. Reaktorerna matades sedan med vatten med olika sammansättning, främst gällande järnhalt. Även befintlig data för verket och uppgifter kring förutsättningarna på andra reningsverk med en fungerande biologisk fosforreduktion undersöktes. Pilotförsöket visade att det går att tvätta bioslammet på järn då en sjunkande halt sågs under försökets gång. Halten sjönk från 40 mg Fe/g TS till 18 mg Fe/g TS i försöksreaktorn. En fungerande fosforrening uppnåddes aldrig så inga slutsatser gällande besparingar, eller vid vilka järnhalter en fungerande rening sker, kan dras. Andra reningsverk med biologisk fosforrening har kring 10 mg Fe/g TS vilket ger en indikation på vad halten bör vara. Pilotförsöket visade också att dosering av polymer ledde till att stora delar av den inkommande kolkällan fälldes, kolkälla som behövs för fungerande fosfor- och kväverening. Recirkulation av nitratkväve sågs hämma det fosforsläpp som vid fungerande rening ska ske i den anaeroba zonen och tros ha stört reningen under försökets gång. Förutsättningarna för biologisk fosforrening på Kungsängsvrket anses inte vara optimala gällande avloppsvattnets sammansättning, recirkulering av nitratkväve till den anaeroba zonen och mängden lättillgänglig kolkälla från hydrolysbassängen. / Phosphorus is an essential element but can cause eutrophication when present in high concentrations. Emission requirements from municipal wastewater treatment plants are therefore strict. Today chemical precipitation is common but there are advantages to using a biological method. It is based on creating conditions that favor growth of a special type of bacteria. These bacteria absorb more phosphorus than they need for growth. To do this they need alternating anaerobic and aerobic zones and access to carbon and phosphorus. A combination between the two methods are common but the precipitation chemicals can under some conditions disturb the biological removal.   At Kungsängsverket the process of biological phosphorus removal has been in place since 2010. It has not worked adequately and the reason could be high concentrations of iron in the biological sludge. The purpose of this thesis has therefore been to investigate whether it is possible to wash out the iron from the bio-sludge and as a result reach a satisfying reduction of phosphorus, to see at which iron content this might happen and what kind of savings a functioning biological phosphorus removal might lead to for Uppsala Vatten och Avfall AB.  To test the hypothesis two reactors were built, a reference reactor and an experimental reactor. The two were fed with water with different compositions, primarily regarding iron content. Also, existing data was examined from the plant and records regarding sludge composition at plants with working biological phosphorus removal. The pilot test showed that it was possible to wash out the iron from the biological sludge. Iron content in the experimental reactor went down from 40 mg Fe/g DM to 18 mg Fe/g DM. A satisfying reduction of phosphorus was never achieved and no conclusions can be drawn regarding savings or at which iron content a reduction might happen. Other wastewater treatment plants with biological phosphorus reduction have shown to have a content of about 10 mg Fe/g DM which can be used as an indication. According to the pilot test dosing of polymer can lead to a large precipitation of carbon source. Lack of carbon will inhibit phosphorus and nitrogen removal. Circulation of nitrate repressed the release of phosphate in the anaerobic zone and is believed to have disturbed the removal during the pilot. The conditions for biological phosphorus removal at Kungsängsverket are not ideal as to the composition of the wastewater, the circulation of nitrate to the anaerobic zone and the amount of carbon source from the hydrolysis.

<p>Waste Stabilisation Ponds (WSPs) are characterised by low phosphorus (P) removal capacity. Heterotrophic bacteria are principal microbial agents in WSPs in addition to algae. As treatment proceeds in WSPs, algal growth increases and pH rises, this has lead to believe that P removal is mainly through sedimentation as organic P algal biomass and precipitation as inorganic P. In activated sludge treatment plants (AS), microbial P removal has been improved and is termed as enhanced biological phosphorus removal. There was a need to establish whether it was possible to enhance P removal in WSPs. A performance assessment of pond system at the University of Dare s Salaam (UDSM), Tanzania, has shown that 90% of the P removed was in the primary pond (facultative) and the rest in the maturation pond (aerobic).</p><p>In these studies, a pure strain A. hydrophyla was isolated from an activated sludge wastewater treatment plant in Sweden. This plant has a train that functions with enhanced biological phosphorus removal. The strain was tested for P uptake in minimal media supplemented with glucose, succinate or acetate, grown aerobically and anaerobically/aerobically. This strain was able to take up P without having been subjected to the anaerobic phase. It was observed that P uptake was enhanced after the anaerobic phase with media supplemented with glucose, but not with succinate or acetate. Phosphorus uptake repeatedly followed the bacterial growth pattern with correlation coefficients of more than 95%. Therefore P removal has a direct correlation with bacterial growth.</p><p>Two isolates Acinetobacter sp. (isolated from the primary facultative pond) and E .coli (isolated from the maturation pond) were obtained from a tropical WSP treatment system at the UDSM. They were subjected to aerobic P uptake experiment similar to those of A.hydrophyla. The uptake per unit absorbance of bacterial growth was found to be comparable to that of A.hydrophyla, isolated from AS. These results showed that heterotrophic activity is important in WSPs. It is possible to enhance P removal in these systems by designing the primary ponds for maximum heterotrophic activity and probably enrichment.</p>

The laboratory-scale University of Cape Town (UCT) process was designed to investigate the effects of changing COD/TP ratios on the performance of biological nutrient removal (BNR) processes. Specific objectives of the research were to investigate the effects of COD/TP ratio on the rates of phosphorus removal, COD removal, nitrogen removal, PHB utilization and oxygen uptake. The system was fed with municipal wastewater and operated at 20° C. The influent COD concentration was held approximately constant while the phosphorus concentration was varied to obtained the desired COD/TP ratio. Once robust enhanced biological phosphorus removal (EBPR) has been established, the COD/TP ratios of 20, 30, 40 and 60 were investigated. The COD/TP ratio of the influent wastewater was observed to have a substantial effect upon the performance of the UCT BNR system. The amount of phosphorus removed by the system and the percent phosphorus in the aerobic zone MLVSS decreased as the COD/TP ratio increased. In addition, the amount of phosphorus released in the anaerobic zone per unit of COD removed in the anaerobic zone increased as the COD/TP ratio decreased. From this research, the amount of anaerobic COD removal required to remove 1 mg/L of phosphorus in the aerobic zone approached a minimum value as the COD/TP ratio decreased. It was also shown that PHB production increased as the COD/TP ratio increased. The highest specific oxygen uptake rate was always observed in the second aerobic reactor and tended to increase as the COD/TP ratio increased. However, the changes in the COD/TP ratio did not significant affect COD removal, nitrogen removal and the observed yield coefficient, but did strongly affect the MLSS concentration. The MLSS concentration at the COD/TP ratio of 60 was only 55% of that at the COD/TP ratio of 20. A high level of anaerobic COD removal, an elevated percent phosphorus in the waste activated sludge (WAS) and a high soluble effluent phosphorus concentration can be used as indicators that the system is operating under COD limiting conditions. Several phenomena were also observed during this research. Firstly, the performance of the UCT BNR system for EBPR was greatly enhanced by reducing the aerobic volume. Secondly, the correlation between non-oxic phosphorus release and the aerobic phosphorus uptake improved when anoxic phosphorus release was taking into consideration. This indicated that the anoxic phosphorus release was not secondary release once the aerobic zone volume was reduced. Finally, no denitrification was observed in the aerobic zone from this study, based on the assumption that 12% of nitrogen was required for bacterial growth. / Master of Science