Sample preparation and analysis for atmospheric pressure matrix assisted laser desorption ionization time of flight mass spectrometry (AP- MALDI-TOF-MS) was investigated. By investigating the effects that sample preparation has upon MALDI signal, better analysis can be carried out. The influence of sample deposition was studied by not only observing the signal intensity produced but also by quantitation. Isotope dilution mass spectrometry (IDMS) was used for the quantitation of three different analytes. The results indicated that not only was signal greatly affected by sample deposition but the effect on quantitation error was also statistically significant among the three different sample deposition techniques that were evaluated. <br>Components of sample preparation solution were studied using polyethylene glycol (PEG) and polystryrene (PS) of different weights. This study altered the amounts of matrix, analyte and cationizing agent that were used to make up each sample. Not only did the sample signal intensity greatly vary which had statistical significance but a shifting of the polymer sample peaks was also observed. This confirms that sample preparation is of extreme importance for MALDI analysis. <br>Carpet fibers, glutathione and cell wall extracts from the bacteria Staphylococcus Epidermidis were also studied by AP- MADLI-TOF-MS. These analytes were carefully studied to provide an accurate characterization of each. The diversity of the analytes studied highlights the incredible capabilities that MADLI possesses being able to analyze a range of analytes. Though the samples were diverse each one was able to be completely and comprehensively analyzed using AP-MALDI-TOF-MS. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry / PhD / Dissertation

Adhesion of bacteria is a key event in biofilm formation and is mediated by bacterial adhesins recognising host or bacterial partner receptors. In oral biofilm formation, primary Actinomyces and Streptococcus colonizers adhere to salivary pellicle proteins such as proline-rich proteins (PRPs) as well as to mucosal surfaces. Subsequently, Actinomyces and Streptococcus strains and other bacteria, such as Veillonella, Fusobacterium and Porphyromonas, adhere to each other. The nature of this community is highly important for the health or disease status, although specific pathogenic species may also have been implicated. The aim of this thesis was to study key players in early oral colonisation, Actinomyces and Streptococcus species, and more specifically the nature of their adhesins and ligands. A further aim was to study the function of the salivary PRP proteins and an innate peptide derived thereof on bacterial adhesion, proliferation and regulation of pH, i.e. key factors in biofilm formation. In paper I and II, adhesion, proliferation and pH affecting features of the RGRPQ (arginine-glycine-arginine-proline-glutamine) peptide, derived from PRP-1, were demonstrated. By use of an alanine-scan (I), motifs for adhesion inhibition and desorption of Actinomyces naeslundii, and proliferation stimulation, ammonia production and inhibition of sucrose induced pH drop by Streptococcus gordonii were indicated. The RGRPQ peptide also stimulated S. gordonii colonisation in vivo. In paper II, a more sophisticated quantitative structure-activity relationship (QSAR) study, using statistical molecular design (SMD) and multivariate modelling (partial least squares projections to latent structures, PLS), further narrowed down the RGRPQ peptide motifs. The R and Q amino acids were crucial for activity. For proliferation a hydrophobic and large size third position amino acid was crucial, while adhesion inhibition and desorption needed a small hydrophilic second position amino acid. All functions depended on a low polarity hydrophobic fourth position. Accordingly, activities could be optimized separately, with decreased function in the others. In paper III and IV, focus was on the bacterial adhesins and their binding epitopes. The genes for FimA major subunit proteins of type-2 fimbriae were sequenced from A. naeslundii genospecies 1 and 2 and Actinomyces odontolyticus, each with unique carbohydrate binding specificities (III). Three major subtypes of FimA proteins were found that correlated with binding specificity, including a novel fimA gene in A. odontolyticus. All subtypes contained a pilin, LPXTG and E box motif. In paper IV, multiple PRP binding patterns for Actinomyces and Streptococcus strains were mapped using a hybrid peptide construct. The two most deviating binding groups deviated in type-1 fimbriae mediated binding to milk and saliva protein ligands. In conclusion, differences in bacterial adhesins and their ability to utilise salivary proteins may render bacteria tropism for different niches. Peptides derived from protein receptors, such as RGRPQ, may be important modulators of biofilm formation, giving commensal bacteria a competitive edge in the bacterial community.

Sulphide containing streams must be treated before releases to environment due to the toxicity, corrosivity and unpleasant odour of sulphide. Anaerobic chemolithotrophic desulphurization under denitrifying conditions is the preferred process when compared with others like physicochemical processes, photoautotrophic and aerobic chemolithotrophic desulphurizations as the catalysts, high pressure, high temperature, light energy and oxygen are not needed. Another main advantage of this process is that the denitrification can be achieved with desulphurization simultaneously. In this work, the anaerobic chemolithotrophic desulphurization under denitrifying conditions (autotrophic denitrification) and heterotrophic denitrification processes were studied. Desulphurization under denitrifying conditions was studied in continuous stirred tank bioreactors (CSTB), while batch, continuous stirred tank and biofilm reactors were used to investigate the heterotrophic denitrification. The kinetics of desulphurization, autotrophic and heterotrophic denitrifications obtained in different systems and under various conditions were compared.<p> Using three different feed sulphide concentrations in the range 10-20 mM, a linear relationship between sulphide loading rates and sulphide removal rates was observed in continuous stirred tank reactors, regardless of initial sulphide concentration. The highest sulphide removal rate of 1.79 mM h-1 was obtained in CSTB fed with 15 mM sulphide. In these systems cell washout occurred at lower dilution rates as sulphide concentration in the feed was increased from 10 to 20 mM. The ratio of sulphide to nitrate loading rates influenced the composition of the sulphur oxidation end products where higher ratios favored the formation of elemental sulphur and lower ratios promoted the formation of sulphate.<p> In the batch system initial concentration of nitrate (5 to 50 mM) did not have a notable effect on denitrification process. Nitrate was converted to nitrite first and the produced nitrite was then converted to other gaseous end products such as nitrogen. Increases of temperature in the range of 15 to 35ºC increased the bacterial growth rate significantly with the value of apparent activation energy for specific growth rate being 60.6 kJ mol-1. Using the experimental data generated in two continuous bioreactors operated with feeds containing 10 and 30 mM nitrate biokinetic coefficients for heterotrophic denitrification were determined. The values of µm, Ks, ms, YMX/S, kd for initial nitrate concentrations of 10 and 30 mM were 0.087 and 0.082 h-1, 2.01 and 5.27 mM (NO3-), 1.441 and 1.096 mM (NO3-) (g biomass) -1 h-1, 0.011 and 0.013 g (biomass) (mM NO3-)-1, and 0.016 and 0.014 h-1 respectively. In the biofilm system the linear relationship between nitrate loading rate and nitrate removal rate was observed again for the whole range of tested nitrate loading rate range (up to 183 mM h-1), regardless of the approach used to increase the loading rate (increases in feed flow rate or feed nitrate concentration). The highest nitrate removal rate was 183 mM h-1 which was around 194 times higher than that achieved in the continuous stirred tank bioreactor with free cells.<p> A comparison of the autotrophic and heterotrophic denitrification processes studied in the CSTB system indicated that in case of autotrophic denitrification wash-out occurred suddenly and at a much lower loading rate of 0.75 to 0.96 mM (NO3-) h-1 for initial sulphide concentrations 10 to 20 mM, while in case of heterotrophic denitrification increase of nitrate loading rate did not have such a drastic effect and removal rate of nitrate decreased slowly with the increases of nitrate loading rate. A comparison of the kinetic data obtained in the biofilm reactor in the present work and those generated for autotrophic denitrification in an earlier work conducted at University of Saskatchewan (Tang, 2008) showed that the dependency of nitrate removal rate on its loading rate were linear in either case and somewhat similar. However, the maximum nitrate removal rate obtained in the heterotrophic system (183 mM h-1) was much higher than that obtained in the autotrophic system with sulphide.

ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23. / February 2006

MELiSSA (Micro Ecological Life Support System Alternative) és el sistema desenvolupat per l'Agència Espacial Europea (ESA) i el consorci MELiSSA en el camp del suport de vida durant missions de llarga durada a l'espai. Basat en un ecosistema aquàtic, MELiSSA va ser concebut com una eina per desenvolupar la tecnologia necessària per a un sistema de suport de vida biològic que en un futur ha de permetre la producció d'aliment, aigua i oxigen a partir dels residus orgànics generats per una tripulació. Per assolir aquest objectiu, el concepte MELiSSA compta amb l'activitat combinada de cinc compartiments colonitzats per diferents microorganismes i plantes superiors, interconnectats entre ells. Aquesta tesi es centra en el tercer compartiment del bucle MELiSSA, en el qual l'amoni és convertit a nitrat, que és la font de nitrogen més adequada per al creixement dels cianobacteris i plantes superiors que colonitzen els compartiments fotosintètics. L'oxidació biològica d'amoni a nitrat té lloc en dues etapes successives que porten a terme dos tipus de soques bacterianes. En el projecte MELiSSA aquest procés es porta a terme en una columna de llit fix mitjançant Nitrosomonas europaea i Nitrobacter winogradkyi immobilitzats sobre un suport polimèric, i amb aportació d'aire en el mateix sentit de circulació que el medi líquid. El reactor pilot del tercer compartiment ha estat operant a la planta pilot del projecte MELiSSA durant períodes prolongats de temps abans de l'inici del treball realitzat en aquesta tesi. La principal aportació d'aquesta tesi es troba en l'obtenció de nova informació sobre el funcionament del reactor a través d'un estudi detallat de la biopel·lícula i també mitjançant el desenvolupament d'un model matemàtic que ens permetrà estudiar els efectes de diferents paràmetres d'operació sobre el procés i l'estructura de la biopel·lícula. S'implementaran també els aparells de mesura necessaris per millorar la qualitat de la monitorització de les diferents espècies de nitrogen a la fase líquida. Els coneixements adquirits en la realització d'aquest treball seran utilitzats per portar a terme el re-disseny del reactor per tal de millorar-ne el funcionament dins de la planta pilot del projecte MELiSSA. / MELiSSA (Micro Ecological Life Support System Alternative) is the system developed by the European Space Agency (ESA) and the MELiSSA consortium in the field of life support for long term manned missions in Space. Based on the principle of an aquatic ecosystem, MELiSSA was conceived as a tool to develop the required technology for a future biological life support system. Its final aim is the production of food, fresh water and oxygen from the organic wastes of a crew. To achieve this goal, the MELiSSA concept is based on the use of five interconnected compartments colonised by several microorganisms and higher plants. This thesis is focused on the third compartment of the MELiSSA loop, in which ammonium is converted to nitrate, the most suitable nitrogen source for the growth of the bacteria and higher plants colonising the photosynthetic compartment. The biological oxidation of ammonium to nitrate, which consists of two successive reactions carried out by two different bacterial strains, takes place in a packed bed biofilm reactor. Nitrosomonas europaea and Nitrobacter winogradskyi are immobilised on a polymeric support, with air flowing cocurrently with the feed medium. The pilot-scale reactor of compartment III (CIII) had been in operation in the MELiSSA pilot plant for several years before the start of the present work. The main contributions of this thesis are in increasing the understanding of the reactor performance by studying the nitrifying biofilm in depth, and by developing a mathematical model that allows the effects of different operational parameters on the process and on the biofilm structure, to be studied. Moreover, continuous monitoring of the nitrifying efficiency will be improved by installing the necessary on-line equipment to experimentally measure the concentrations of all the nitrogen species in the liquid phase. The additional knowledge achieved on the reactor performance via this work will finally lead to re-design the reactor hardware for optimal performance in the MELiSSA pilot plant. The knowledge acquired in this thesis was finally used to define the main features of the re-design of the pilot reactor of the MELiSSA compartment III.

Sulphide containing streams must be treated before releases to environment due to the toxicity, corrosivity and unpleasant odour of sulphide. Anaerobic chemolithotrophic desulphurization under denitrifying conditions is the preferred process when compared with others like physicochemical processes, photoautotrophic and aerobic chemolithotrophic desulphurizations as the catalysts, high pressure, high temperature, light energy and oxygen are not needed. Another main advantage of this process is that the denitrification can be achieved with desulphurization simultaneously. In this work, the anaerobic chemolithotrophic desulphurization under denitrifying conditions (autotrophic denitrification) and heterotrophic denitrification processes were studied. Desulphurization under denitrifying conditions was studied in continuous stirred tank bioreactors (CSTB), while batch, continuous stirred tank and biofilm reactors were used to investigate the heterotrophic denitrification. The kinetics of desulphurization, autotrophic and heterotrophic denitrifications obtained in different systems and under various conditions were compared.<p> Using three different feed sulphide concentrations in the range 10-20 mM, a linear relationship between sulphide loading rates and sulphide removal rates was observed in continuous stirred tank reactors, regardless of initial sulphide concentration. The highest sulphide removal rate of 1.79 mM h-1 was obtained in CSTB fed with 15 mM sulphide. In these systems cell washout occurred at lower dilution rates as sulphide concentration in the feed was increased from 10 to 20 mM. The ratio of sulphide to nitrate loading rates influenced the composition of the sulphur oxidation end products where higher ratios favored the formation of elemental sulphur and lower ratios promoted the formation of sulphate.<p> In the batch system initial concentration of nitrate (5 to 50 mM) did not have a notable effect on denitrification process. Nitrate was converted to nitrite first and the produced nitrite was then converted to other gaseous end products such as nitrogen. Increases of temperature in the range of 15 to 35ºC increased the bacterial growth rate significantly with the value of apparent activation energy for specific growth rate being 60.6 kJ mol-1. Using the experimental data generated in two continuous bioreactors operated with feeds containing 10 and 30 mM nitrate biokinetic coefficients for heterotrophic denitrification were determined. The values of µm, Ks, ms, YMX/S, kd for initial nitrate concentrations of 10 and 30 mM were 0.087 and 0.082 h-1, 2.01 and 5.27 mM (NO3-), 1.441 and 1.096 mM (NO3-) (g biomass) -1 h-1, 0.011 and 0.013 g (biomass) (mM NO3-)-1, and 0.016 and 0.014 h-1 respectively. In the biofilm system the linear relationship between nitrate loading rate and nitrate removal rate was observed again for the whole range of tested nitrate loading rate range (up to 183 mM h-1), regardless of the approach used to increase the loading rate (increases in feed flow rate or feed nitrate concentration). The highest nitrate removal rate was 183 mM h-1 which was around 194 times higher than that achieved in the continuous stirred tank bioreactor with free cells.<p> A comparison of the autotrophic and heterotrophic denitrification processes studied in the CSTB system indicated that in case of autotrophic denitrification wash-out occurred suddenly and at a much lower loading rate of 0.75 to 0.96 mM (NO3-) h-1 for initial sulphide concentrations 10 to 20 mM, while in case of heterotrophic denitrification increase of nitrate loading rate did not have such a drastic effect and removal rate of nitrate decreased slowly with the increases of nitrate loading rate. A comparison of the kinetic data obtained in the biofilm reactor in the present work and those generated for autotrophic denitrification in an earlier work conducted at University of Saskatchewan (Tang, 2008) showed that the dependency of nitrate removal rate on its loading rate were linear in either case and somewhat similar. However, the maximum nitrate removal rate obtained in the heterotrophic system (183 mM h-1) was much higher than that obtained in the autotrophic system with sulphide.

Differential gene expression of P. aeruginosa in a rhizosphere biofilm on poplar tree roots was examined in order to identify new virulence factors from this human pathogen. Changes in gene expression for poplar trees contacted with P. aeruginosa was examined as well to identify the response of poplar roots to P. aeruginosa infection. This is the first study of the whole-transcriptome analysis of P. aeruginosa on a plant tree root. The 20 most highly-induced genes of P. aeruginosa were examined for their role in biofilm formation, rhizosphere colonization, barley germination, and poplar tree killing assays. Seven previously uncharacterized virulence genes (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150, and PA4295) were identified. The role of PA2663, a hypothetical protein discovered in the microarrays of P. aeruginosa while killing poplar trees, was examined in further detail. Expression of PA2663 protein increases biofilm formation in P. aeruginosa PAO1 drastically. By complementing the PA2663 mutation in trans and by studying with DNA microarrays and RT-PCR the PA2663 mutant vs. the wild-type strain, PA2663 was confirmed to be related to biofilm formation and was found that it is the first protein to control the psl operon in P. aeruginosa PAO1. Furthermore, PA2663 protein increases pyoverdine synthesis and quorum sensing (QS)- regulated phenotypes. A biofilm formation-related hypothetical protein, PA0939, was identified in this study. The effects of indole and 7-hydroxyindole on P. aeruginosa virulence factors were also examined for the first time. Indole and 7HI repressed expression of mexGHI-opmD multidrug efflux pump genes and genes involved in synthesis of QS-regulated virulence factors (pyocyanin, rhamnolipid, PQS, and pyoverdine production). In addition, the effects of an anti-cancer uracil analog, 5-fluorouracil (5-FU) on P. aeruginosa virulence factors and E. coli K-12 biofilm formation were examined. 5-FU repressed biofilm formation, abolished quorum-sensing phenotypes, and reduced virulence in P. aeruginosa. DNA microarray and biofilm studies with 5-FU in E. coli revealed that 5-FU controls biofilm formation through the AriR protein in E. coli K-12 strain. The effects of lsrR and lsrK mutations on E. coli biofilm formation were also examined by flow cell experiments.

Biofilms are highly organized bacterial structures that are attached to a surface. They are ubiquitous in nature and may be detrimental, causing numerous types of illnesses in living organisms. Biofilms in the human oral cavity are the main cause of dental caries and periodontal diseases and can act as a source for pathogenic organisms to spread within the body and cause various types of systemic diseases. Streptococcus mutans is the primary etiological agent of dental caries, the single most chronic childhood disease. In many cases, quorum sensing (QS) is required for initial formation and subsequent development of biofilms and the signaling molecule autoinducer 2 (AI- 2) has been well studied as an inter-species QS signaling molecule. However, recent reports also suggest that AI-2-mediated signaling is important for intra-species biofilm formation in both Gram-negative and positive bacteria. Therefore, there is significant interest in understanding the role of different QS signals such as AI-2 in oral biofilm formation. Microfluidic devices provide biomimetic environments and offer a simple method for executing multiple stimuli experiments simultaneously, thus, can be an extremely powerful tool in the study of QS in biofilms. In this study, we report conditions that support the development of S. mutans biofilms in microchannel microfluidic devices, and the effects of extracellular addition of chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione (DPD; precursor of AI-2) on mono-species S. mutans luxS (AI-2 deficient strain) biofilm formation using a gradient generating microfluidic device. S. mutans wild type (WT) and luxS biofilms were developed in nutrient rich medium (25% brain heart infusion medium, BHI + 1% sucrose) for up to 48 h. Maximum biofilm formation with both strains was observed after 24 h, with distinct structure and organization. No changes in S. mutans luxS biofilm growth or structure were observed upon exposure to different concentrations of AI-2 in a gradient generating device (0 to 5 M). These results were also validated by using a standard 96-well plate assay and by verifying the uptake of AI-2 by S. mutans luxS. Our data suggest that extracellular addition of AI-2 does not complement the luxS deletion in S. mutans with respect to biofilm formation.

Bacterial biofilms form on liquid/air and liquid/solid surfaces and consist of cells combined with an extracellular matrix such as exopolysaccharides, extracellular DNA, and glycoproteins. Bacteria have up to a 1000-fold increase of antibiotic resistance in biofilms compared to planktonic cells. Furthermore, biofilm cells show better tolerance to adverse environmental conditions such as nutrition limitations, temperature changes, pH changes, and non-optimal osmotic conditions. In Escherichia coli, the outer membrane protein OmpA increased biofilm formation on polystyrene, polypropylene, and polyvinyl chloride surfaces while it decreased biofilm formation on glass surfaces. This surface-dependent phenotype was because OmpA inhibits cellulose production by inducing the CpxRA two-component signal transduction pathway, and cellulose inhibits biofilm formation on plastic due to its hydrophilic nature. We discovered, and then engineered, BdcA (formerly YjgI), for biofilm dispersal. We found that in E. coli, BdcA increases motility and extracellular DNA production while it decreases exopolysaccharide production, cell length, and aggregation. We reasoned that the 3, 5-cyclic diguanylic acid (c-di-GMP) levels increase upon deleting bdcA, and showed that BdcA binds c-di-GMP in vitro. In addition, we used protein engineering to evolve BdcA for greater c-di-GMP binding and found that the single amino acid change E50Q causes nearly complete biofilm dispersal. We isolated Proteus mirabilis from the blowfly Lucilia sericata, which swarmed significantly. By motility screening and complementation with putative interkingdom signal molecules that have been shown to attract flies, we found lactic acid, phenol, NaOH, KOH, putrescine, and ammonia restore the swarming motility of seven different swarming deficient mutants. These mutants and putative signal molecules will be further tested for fly attraction and oviposition. Acetylation of lysine residues is conserved in all three kingdoms although its role in bacteria is not clear. We demonstrated that acetylation enables E. coli to withstand environmental stresses. Specifically, the bacteria became more resistant to heat and oxidative stress. Furthermore, we showed that the increase in oxidative stress resistance is due to the induction of catalase gene katG. Hence we demonstrate for the first time a specific physiological role for acetylation in prokaryotes.

Rent vatten är en förutsättning för allt liv på jorden men utsläpp av föroreningar från mänskliga aktiviteter snedvrider ekosystemen med allvarliga konsekvenser som följd. Bristfällig rening och utsläpp av obehandlat avloppsvatten till naturen orsakar syrebrist och övergödning i vattenmiljöer. I svenska städer renas avloppsvatten i kommunala reningsverk genom mekanisk, biologisk och kemisk rening. Utanför städerna där kommunal anslutning inte är möjlig finns små anläggningar, så kallade enskilda avlopp, som renar avloppsvatten från en enskild fastighet eller ett mindre antal hushåll. Avloppsvatten från bensinstationer renas ofta i enskilda avloppsanläggningar där små reningsverk blir en allt vanligare reningsmetod. Reningsverken dimensioneras utifrån en branschgemensam definition av hushållsavlopp, vilken kan avvika starkt från det avloppsvatten som produceras i fastigheten till följd av avsaknad av bad-, disk- och tvättvatten. Framförallt är höga halter av organiskt material och kväve orsaker till skillnaderna. Naturvårdsverkets krav för utsläpp från enskilda avlopp förväntas vara uppfyllda även för avloppsvattnet från bensinstationer, trots att förutsättningarna i avloppet skiljer sig avsevärt från de som ligger till grund för kraven. I utredningen ingår tre små reningsverk som renar avloppsvatten med höga halter organiskt material från bensinstationer. De två reningsverken med biofilmsprincip har problem att klara kraven för rening av kväve. Dessutom undersöks inverkan av en reservoar och hypotesen är att den genomsnittliga reningsgraden ökar då reservoaren utjämnar de starkt varierande flödena. I reningsverket med aktivt slam uppstår problem med att det aktiva slammet dör och avger fräna lukter. Prover togs och analyserades vid samtliga reningsverk. Utredningen syftar till att finna orsakerna bakom de nedsatta reningsgraderna och föreslå åtgärder. De frågor som ställs är hur kvävereningen fungerar i biofilmsanläggningarna och hur den kan förbättras samt vad orsakerna är till varför det aktiva slammet dör och hur det kan undvikas. Resultaten pekade på fosforbrist i reaktorerna på biofilmsanläggningarna, vilket hämmade mikroorganismernas aktivitet vid reduktion av organiskt material som försvårade möjligheterna till kvävereduktion. Storleken på tankarna var sannolikt för små eller för få i förhållande till den höga organiska belastningen. Reservoaren i biofilmsanläggningen hade enligt utredningen en utjämnande effekt på reningsgraden av kväve och fosfor, men hade ingen påverkan på reduktionen av organiskt material. I aktivslamanläggningen orsakade den höga halten organiskt material syrebrist, vilket tillsammans med sulfat från fällningskemikalien ledde till bildandet av illaluktande vätesulfid samt det giftiga och svarta järnsulfid. För att öka reningsgraden av kväve i biofilmsanläggningarna föreslås att fosforreduktionen placeras efter den biologiska reningen och tillsats av en annan typ av flockningsmedel i slamavskiljaren rekommenderas för att underlätta reduktionen av organiskt material. För att undvika sulfidbildning i aktivslamanläggningen krävs en ökad syresättning. En kloridbaserad fällningskemikalie förordas framför en sulfatbaserad, eftersom tillgång till sulfat möjliggör bildandet av sulfider. / Clean water is essential for all life but emissions of pollutants from human activities are disturbing the balance in ecosystems leading to severe consequences. Poor treatment of domestic wastewater can cause anoxic waters and eutrophication in aquatic environments. In Swedish cities, domestic wastewater is treated in municipal wastewater treatment plants through mechanical, biological and chemical treatments. Where connection to the municipal wastewater treatment plants is not available, the wastewater from a single or a few households are treated in decentralized treatment plants. Wastewater, mainly from toilets, on gas stations is a typical example of individual sewage and decentralized treatment plants are becoming more common. The decentralized treatment plants are dimensioned based on an industry-wide definition of domestic sewage, which can differ greatly from the sewage produced in gas stations because of the lack of diluting bathing and washing water. The high concentration of organic matter and nitrogen is mainly the explanation to the differences. Regulation for emissions from domestic wastewater that has been made by the Swedish Environmental Protection Agency applies to this special wastewater even though the preconditions in the wastewater are significantly different from the ones that form the basis for the requirements. This study includes three decentralized treatment plants treating domestic wastewater with a high organic load having problems to meet the requirements of the authorities. Two of the treatment plants are based on the principle of fixed biofilm and is investigated because of their inadequate reduction of nitrogen. The difference between the two plants is a reservoir and a hypothesis is that it has a positive effect of the total reduction rate by smoothing of the incoming flow. The third treatment plant is a batch reactor with activated sludge that now and then has problems with pungent odors and dead activated sludge colored black. Samples were taken and analyzed at all treatment plants. The questions posed are how the reduction of nitrogen is working in the biofilm plants and how it can be improved, as well as the question about the reasons behind the dead sludge in the batch reactor with activated sludge and how it is prevented. The effect of the reservoir on the reduction rate is also investigated. The results revealed a lack of phosphorus in the bioreactors with fixed biofilm, which inhibited the microbial activity and thus the reduction of organic matter and nitrogen. The size of the reactors was at the same time probably too small relative to the organic load. The investigation showed that the reservoir in the biofilm plant had a positive effect on the reduction rate for total nitrogen and phosphorus. The high concentration of organic matter caused poor oxygen exchange in the batch reactor with activated sludge, leading to the formation of toxic hydrogen sulfide and iron sulfide. The sulfides explained the bad odors and why the activated sludge died. The proposed action for the biofilm plant is to place the reduction of phosphorus after the biological treatment, for example filtration. In the batch reactor with activated sludge is increased oxygen exchange suggested, in combination with a new precipitation chemical that is not based on iron sulfate, but chloride, because access to sulfate enables the formation of sulfides.