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.

Phosphorus removal in wastewater treatment is commonly achieved by chemical precipitation, enhanced biological phosphorus removal (EBPR) or a simultaneous combination of the two. A defined anaerobic condition is widely regarded as the critical element to sustain EBPR. However, this study demonstrates that EBPR is indeed occurring in a 4.5 m3/h membrane bioreactor (MBR) pilot plant without a defined anaerobic condition. Although designed for chemical precipitation alone, a low average Fe/P molar ratio (iron dose/phosphorus removed) of 0.9 ± 0.1 suggests that EBPR could be contributing to a simultaneous phosphorus removal. Weekly flow-proportional grab samples through the process showed a phosphate (P) release between the primary anoxic tanks, followed by a larger uptake in the aerobic tanks. In laboratory batch tests with limited acetate supply in the presence of nitrates, the anoxic P-release began and then abruptly stopped whilst the nitrate concentration continued to decrease. This could be explained by denitrifiers out-competing PAOs for soluble substrate since a large P-release occurred when excess acetate was supplied in the presence of nitrates. It is therefore unlikely that PAOs are operating in the pilot despite the presence of nitrates as was concluded in a study with similar spontaneous EBPR observations. Instead, it is suggested that EBPR is enabled by intermittent anaerobic conditions in the primary anoxic tanks due to low nitrate concentrations (< 1 mg NO3/l) recirculating back after post-denitrification. The external carbon source supplied to the pilot was changed from glycerol to ethanol to assess any effect on the spontaneous EBPR. After ethanol had been supplied for 30 days, increased P-release rates were observed in batch tests supplied with ethanol (0.1 to 0.4 mg P/g VSS∙h) and batch tests supplied with acetate (8.6 ± 0.4 to 10.3 ± 0.4 mg P/g VSS∙h). However, the overall consumption of glycerol was less than that of ethanol, whilst the total phosphorus removal and the Fe/P ratio remained similar whether ethanol or glycerol was supplied to the pilot plant. Should operators wish to avoid any possible spontaneous P-release in the post-denitrification step, methanol is recommended as the external carbon source when considering laboratory P-release results, past research and operation costs. / Fosforrening av avloppsvatten uppnås vanligtvis genom kemisk utfällning, enhanced biological phosphorus removal (EBPR) eller en kombination av dessa två samtidig. Ett definierat anaeroba förhållanden betraktas allmänt som det kritiska elementet för att upprätthålla EBPR. Dock visar denna studie att EBPR verkligen förekommer i en pilotanläggning med membranbioreaktor (MBR) utan ett definierat anaeroba förhållanden. Veckovis flödesproportionella stickprover genom processen visade en ökad fosfat (P) halt mellan de primära anoxisk tankarna, följt av ett upptag i de aerobiska tankarna. Även om det är planerat för bara kemisk fosforrening, ett lågt medel Fe/P molförhållande (järndos/fosfor bortagning) på 0,9 ± 0,1 föreslår att EBPR kan bidra till ett samtidigt fosforrening. Laboratorie-P-utsläppningstest bekräftade EBPR-aktivitet med en hög medel P-utsläpp av 9,3 ± 0,9 mg P/g VSS∙h med acetat. Tillsammans indikerar dessa resultat att biologiskt fosforrening kompletterade den kemiska fosforrening under undersökningsperioden. P- utsläppningstest visade att närvaron av nitrater inte påverkade PAO-aktiviteter när acetat tillfördes i överskott. Med begränsad acetattillförsel förhindrade emellertid närvaron av nitrater någon initial P-utsläpp och inga efterföljande fermenteringsbiprodukter observerades. Slutsatsen dras att effektiv nitrifikation-denitrifikation och hög recirkulation resulterar i låga nitrathalt i primär anox tankar. Detta orsakar intermittenta anaerobisk förhållanden som, tillsammans med lämpliga substrathalt i inloppsvatten, möjliggör EBPR att ske. Den externa kolkällan som levererades till MBR-piloten ändrades från glycerol till etanol för att undersöka potentialen att öka EBPR-andelen av fosforrening. P-utsläpp i labbtester med etanol dosering ökade från 0,1 till 0,4 mg P/g VSS∙h och tester med acetat dosering ökade från 8,6 ± 0,4 till 10,3 ± 0,4 mg P/g VSS∙h när slam hade anpassats till etanol i 30 dagar. Vid pilotanläggningens drift konsumerades i alla fall mindre glycerol under dess användningsperiod än etanol, och Fe / P- förhållandet var likadant oavsett om glycerol eller etanol tillsatts. Olika andra kolkällor testades i parallella P-utsläppningstest. VFA producerat genom jäsning av matavfall och primärt slam i ett pågående projekt, och huvudsakligen bestående av kapronsyra, resulterade i den näst högsta P- utsläpp. P-utsläpp från alkoholer var märkbart lägre än VFA-baserade kolkällor. Det betyder att risken är låg för oväntat P-utsläpp i den slutliga biologiska zonen om en alkoholbaserad kollkällan doseras där. Minskad förluftning och en liten dos VFA-baserat substrat i den första biologiska zonen skulle bidra till en betydande EBPR som skulle möjliggöra en ytterligare reducerad basdos av järnsulfat.

Over the last two decades, livestock operations have become highly concentrated due to growing trends towards larger, more confined facilities and a decrease in cropland on smaller farms. This has led to greater amounts of excess manure nutrients on farms, increasing the potential for nutrient pollution of water bodies from runoff. The purpose of this study was to determine if enhanced biological phosphorus removal (EBPR) is a viable alternative for managing excess manure nutrients on dairy farms. Assessment of EBPR involved the investigation of various aspects of wastewater treatment modeling and design and farm nutrient management. The fermentation potential (volatile fatty acid (VFA) production) of dairy manure was determined through laboratory analysis to be 15.3% of the total COD. Total VFA production was composed of 57, 23, and 20% acetic, propionic, and butyric acids, respectively. The EBPR component of the BioWin wastewater treatment model was evaluated through a sensitivity analysis. The parameters to which effluent phosphate (PO4) concentration was most sensitive were maximum specific growth rate, growth yield, aerobic PO4 uptake rate per unit poly-b-hydroxybutyrate (PHB) utilized, PHB yield from VFA, PO4 release per unit VFA uptake, and fraction of releasable PO4. An EBPR sequencing batch reactor (SBR) was designed for a dairy farm with 700 lactating cows and 325 ha of corn silage. An economic analysis of EBPR for dairy farms employing P-based manure applications was completed. The cost of hauling excess manure to nutrient deficient farms was the most significant expense in comparing costs of manure management with and without EBPR. For a herd of 700 lactating cows, utilizing EBPR was more economical for farms with 270 ha or less cropland, while EBPR did not offer an economic advantage for farms over 270 ha. / Master of Science

Enhanced biological phosphorus removal (EBPR) has been widely used in municipal wastewater treatment, but no previous studies have examined the application of EBPR to treat dairy manure. This study was conducted to evaluate the (i) performance of pilot-scale EBPR systems treating liquid dairy manure, to balance the ratio of nitrogen to phosphorus in manure to meet crop nutrient requirements, (ii) effects of dissolved oxygen and solids retention time on the efficiency of EBPR, and (iii) effectiveness of gravity thickening for reducing the volume of harvested EBPR aerated mixed liquor. Two pilot-scale EBPR systems were used in this study. The ratio of the manure chemical oxygen demand expressed as volatile fatty acids to phosphorus used ranged from 18:1 to 45:1. The phosphorus removal efficiencies of the EBPR system were investigated at three solids retention times (4, 6 and 10 d), and three dissolved oxygen levels (3, 4 and 6 mg O₂/L). The total phosphorus removal was highest (84%) at 10 d solids retention time and lowest (63%) at 4 d solids retention time. The sludge from the 6 d solids retention time tests had better sludge settling characteristics with a sludge volume index of 62 mL/g compared to 80 mL/g for the 4 d solids retention time. The EBPR system achieved 90% dissolved reactive phosphorus removal when the system was operated at 4 mg O₂/L, and the ratio of nitrogen to phosphorus in effluent increased to about 5:1, which was higher than the normal ratio in dairy manure. On the other hand, phosphorus removal performance deteriorated when dissolved oxygen level was 3 mg O₂/L. In the gravity thickening tests, 93-95 % total suspended solids (TSS) was removed from the settled supernatant, with 1.2 to 1.54 % total solids (TS) in the settled solids after 90 min gravity-induced thickening. The extent of phosphorus release during gravity thickening process needs to be further investigated. / Master of Science

I Sverige förekommer strikta krav på fosforrening av avloppsvatten och detta har bidragit till att kemisk fällning har kommit att dominera som reningsmetod för fosfor vid svenska avloppsreningsverk. Fällningskemikalier är dyrt för reningsverken att köpa in och ger negativ påverkan på miljön vid tillverkning och transport. Strängare reningskrav har ökat behovet av nya reningsmetoder som på ett effektivt och miljövänligt sätt kan rena avloppsvatten från näringsämnen utan att kostnaderna för reningen blir för stora. Biologisk fosforavskiljning (bio-P) utnyttjar mikroorganismer som naturligt kan ackumulera fosfor i sina celler. En kritisk faktor för en väl fungerande biologisk fosforavskiljning är tillgången till kolkälla i form av flyktiga fettsyror (VFA). Kommunalt avloppsvatten innehåller vanligen för lite VFA för att tillgodose bio-P-processens behov. Den totala tillgången på organiskt material i avloppsvattnet är dock ofta stor och möjligheten finns därmed att genom biologisk slamhydrolys internt producera VFA. Vid biologisk hydrolys av slam utnyttjas mikroorganismers naturliga förmåga att under anaeroba förhållanden bryta ned organiskt material till mer lättomsättliga föreningar. Duvbackens avloppsreningsverk i Gävle tillämpar biologisk fosforavskiljning och producerar VFA till bio-P-processen genom primärslamshydrolys och sidoströmshydrolys av returslam. I detta examensarbete har hydrolysprocesserna vid reningsverket utvärderats med avseende på processernas förmåga att producera VFA till det biologiska reningssteget. Utvärderingen gjordes genom att mäta förändringen i halten organiskt material över hydrolysbassängerna samt genom att bedöma effektiviteten i bio-P-processen i fullskala och vid labskaleförsök. Möjligheten att stänga av primärslamshydrolysen vid verket undersöktes. Ett försök till att optimera driften av sidoströmshydrolysen gjordes genom att utföra hydrolysförsök i laboratorieskala. Resultaten indikerade att produktionen av VFA i primärslamshydrolysen var begränsad och att processen därmed bör kunna stängas av. Innan detta görs bör dock kompletterande mätningar av halten löst COD över primärslamshydrolysen utföras. Vid samtliga mättillfällen uppmättes en betydande ökning i halten organiskt material över sidoströmshydrolysen. Baserat på detta och den i examensarbetet konstaterade effektiviteten i bio-P-processen drogs slutsatsen att sidoströmshydrolysprocessen vid Duvbackens reningsverk var välfungerande. Hydrolysförsöken pekade på att ett större utbyte av VFA skulle erhållas om en kortare uppehållstid än den nuvarande användes i sidoströmshydrolysen. Detta bör vidare undersökas genom fullskaleförsök vid verket. / The strong regulations concerning phosphorus removal from wastewater in Sweden has resulted in chemical precipitation being the most common treatment method for phosphorus at Swedish wastewater treatment plants (WWTP). Precipitation chemicals are expensive and have a negative environmental impact when produced and transported. More stringent wastewater treatment requirements have increased the need of new, eco-friendly treatment methods that effectively can remove nutrients from wastewater without being too expensive. Enhanced biological phosphorus removal (EBPR) utilizes microorganisms that have the natural capability of accumulating phosphorus in their cells. A critical factor for a well-functioning EBPR-process is the availability of carbon source in form of volatile fatty acids (VFA). Municipal wastewater normally contains too small amounts of VFA to satisfy the need of the EBPR-process. The total amount of organic matter in the wastewater is on the other hand large and the possibility consequently exists to internally produce VFA through sludge hydrolysis. Biological sludge hydrolysis takes advantage of the natural capability of microorganisms to degrade complex organic compounds into easily degradable organics. Duvbacken WWTP in Gävle uses EBRP for phosphorus removal and produces carbon source through hydrolysis of primary sludge and sidestream hydrolysis of return sludge. In this master thesis the hydrolysis processes at the WWTP was evaluated with regard to the capacity of the processes to produce VFA to the biological treatment step. The evaluation was performed by measuring the change in organic material content over the hydrolysis basins and by estimating the effectiveness of the EBPR-process in full scale and by laboratory experiments. The possibility to turn off the primary sludge hydrolysis process was examined. An attempt to optimize the operation of the sidestream hydrolysis process was made by conducting hydrolysis experiments in laboratory scale. The results indicated that the production of VFA by primary sludge hydrolysis was limited and that it thus would be possible to turn off the process. Before this is done complementary measurements of COD levels over the primary hydrolysis basin should be performed. At all times considerable increments in COD content was measured over the sidestream hydrolysis basin. Based on this and the in the thesis confirmed effectiveness of the EBRP-process the conclusion was drawn that the sidestream hydrolysis of return sludge at Duvbacken WWTP was well functioning. The hydrolysis experiments indicated that a larger VFA yield would be obtained if a shorter retention time than the present was used in the sidestream hydrolysis process. This should further be investigated by experiments in full scale at the WWTP.

The Enhanced Biological Phosphorus Removal (EBPR) process is characterized as the most sustainable process to remove phosphorus from wastewater albeit with high variability in performance efficiency. Thus, unpredictable upsets in the EBPR system is the norm across several wastewater treatment plants throughout Sweden, forcing the hand of the operators to dose higher volume of chemicals to reach the effluent requirements. As future effluent requirements are getting stricter and since higher chemical usage is environmentally and economically unsustainable, this investigation was setup to evaluate which environmental, operational and/or wastewater characteristics correlate with EBPR upsets at full-scale wastewater treatment plant (WWTP), more specifically at Lundåkra WWTP operated by Nordvästra Skånes Vatten och Avlopp (NSVA). The data used in the investigation was collected between 1St January 2018 and 31St December 2020 for a vast number of parameters known to play a key role in biological phosphorus removal. Online sensors as well as external and internal analysis contributed to the data which included parameters such as ‘Total flow at the plant’, ‘pH of the incoming water’, ‘Temperature in aeration basins’, ‘Dissolved oxygen (DO) levels in aeration basins’, ‘Nitrate in aeration basins’, ‘Sludge content in aeration basins’, etc. Other relevant parameters such as ‘Hydraulic retention time (HRT) in the treatment units’, ‘Sludge retention time (SRT) in aeration basin’, ‘Organic loading rate (OLR)’, etc. were calculated. Before the start of this investigation, the two possible explanations were presumed and they can be classified as: (i) upsets as a result of unsuitable environmental conditions and/or error in the operational strategy at the plant and (ii) upsets as a result of toxicity from higher concentration of metals in the influent specifically. Traditional statistical methods such as the t-distributed Stochastic Neighbor Embedding (t-SNE), Spearman Rank Correlation and Principal Component Analysis were used for the purpose of this study to test the first presumed explanation. The t-SNE plot showed that the upsets did not cluster into one large group but instead clamped up into smaller groups scattered across the length of the scale in both dimensions. This points towards the multivariate dependency of the EBPR process and exhibits that upsets might occur even with an operational strategy that produces good results otherwise. This, in turn, eludes to the fact that a non-included parameter such as the ‘daily metal concentrations in the influent’ could be responsible for some or all of the upsets. The Principal Component Analysis (PCA) plot, although noisy, offered an improvement strategy built around the key variables namely ‘nitrate in aeration basin 1 & 2’, ‘sludge content in aeration basin’, ‘SRT in aeration basin’, ‘O2 in aeration basin 1 & 2’ and ‘pH of incoming water’. Therefore, it is recommended that an improvement strategy be devised around them. Multiple causal factors increase the complexity of the analysis by decreasing the correlation coefficients, however, incorporation of the scatterplots presents a clearer picture. The parameters ‘nitrate in aeration basin 1 & 2’ and ‘sludge content in aeration basin’ showed the strongest correlation with phosphate values at the end of biological treatment at -0.32 and 0.42 respectively. The results also open the door to future research and provide direction for further investigations. / Den förbättrade biologiska fosforborttagningsprocessen karakteriseras som den mest hållbara processen för att avlägsna fosfor från avloppsvatten om än med stor variation i prestandaeffektivitet. Således är oförutsägbara störningar i systemet för förbättrad biologisk fosforavskiljning (EBPR) normen bland flera avloppsreningsverk i hela Sverige, vilket tvingar operatörerna att dosera högre volymer kemikalier för att nå avloppskraven. Eftersom framtida avloppskrav blir allt strängare och eftersom högre kemikalieanvändning är miljömässigt och ekonomiskt ohållbar, gjordes denna undersökning för att utvärdera vilka miljö-, drifts- och/eller avloppsvattenegenskaper som korrelerar med EBPR- störningar vid fullskaligt avloppsreningsverk. Närmare bestämt vid Lundåkra reningsverk som drivs av Nordvästra Skånes Vatten och Avlopp. Datan som användes i undersökningen samlades in mellan 1:a januari 2018 och 31:a december 2020 för ett fast antal parametrar som är kända att spela en nyckelroll vid borttagning av biologiskt P. Onlinesensorer samt externa och interna analyser bidrog till datan vilken inkluderade parametrar som 'Totalt flöde vid anläggningen', 'pH för det inkommande vattnet', 'Temperatur i luftningsbassänger', nivåer av upplöst syre (DO) i luftningsbassänger ',' Nitrat i luftningsbassänger ',' Slamhalt i luftningsbassänger ', etc. Andra relevanta parametrar som 'Hydraulisk retentionstid (HRT) i behandlingsenheterna ',' Slamretentionstid (SRT) i luftningsbassäng ',' Organisk belastningshastighet (OLR) ', etc. beräknades. Innan denna undersökning påbjörades antogs de två möjliga förklaringarna och de kan klassificeras som: (i) störningar till följd av olämpliga miljöförhållanden och/eller fel i driftstrategin vid anläggningen och (ii) störningar till följd av toxicitet från högre koncentration av metaller i inflödet specifikt. Traditionella statistiska metoder såsom t- Distributed Stochastic Neighbor Embedding (t-SNE), Spearman Rank Correlation och Principal Component Analysis användes i denna studie för att testa den första förmodade förklaringen. t-SNE- diagrammet visade att störningarna inte samlades i en stor grupp utan istället klämdes ihop i mindre grupper utspridda över skalans längd i båda dimensionerna. Detta pekar mot EBPR-processens multivariata beroende och visar att störningar kan uppstå även med en operativ strategi som annars ger bra resultat. Detta i sin tur undviker det faktum att en icke-inkluderad parameter som "dagliga metallkoncentrationer i inflödet" kan vara orsaken för några eller alla störningar. Principal Component Analysis (PCA)-diagrammet, trots att det var bullrigt, möjliggjorde en förbättringsstrategi byggd kring nyckelvariablerna, nämligen 'nitrat i luftningsbassäng 1 & 2', 'slamhalt i luftningsbassäng', 'SRT i luftningsbassäng', 'O2 i luftningsbassäng 1 & 2' och 'pH av inkommande vatten'. Därför rekommenderas att en förbättringsstrategi utarbetas kring dem. Flera kausala faktorer ökar komplexiteten i analysen genom att minska korrelationskoefficienterna, men spridningsdiagrammen ger en tydligare bild. Parametrarna ‘nitrat i luftningsbassäng 1 & 2’ och ‘slamhalt i luftningsbassäng’ visade starkast samband med fosfatvärden vid slutet av biologisk behandling vid -0,32 respektive 0,42. Resultaten lämnar dörren öppen för framtida forskning och kan vägleda vidare undersökningar.