Syntrophic acetate oxidizing bacteria convert acetate into hydrogen and carbon dioxide and through the mutualistic syntrophic partnership with methanogens the products are further converted to methane in biogas processes operating at high ammonia concentrations. There is very little known about SAOBs, only five have been characterized and had their genome analyzed. The aim of this project was to gain further knowledge about the methane producing pathway of SAOBs with a proteomic approach. Proteins were extracted from biogas sludge with a phenol-based approach and trypsin digestion and peptide recovery were performed using the Suspension Trapping method. Measurement of the peptide content was made with LC-MS/MS. The peptide profiles obtained were screened for the proteins expressed of the mesophilic SAOB Syntrophaceticus schinkii. The data supports earlier suggestions that it utilizes the Wood-Ljungdahl pathway for hydrogen production. Furthermore, the peptide profile revealed that enzymes for the glycine reductase complex and the glycine cleavage system were expressed during high ammonia concentration, indicating a potential role of these enzymes in the methane producing pathway. However, due to partial failure of the sample preparation for mass spectrometry measurements no quantification conclusions could be made. A discussion on how to further improve sample preparation methods as well as how to access the proteome to a large extent is presented.

Syntrophic relationships between fatty acid degrading bacteria and hydrogenotrophic methanogens are important for a well-functioning anaerobic degradation process during biogas production from organic waste material. This is particularly important in biogas processes fed protein-rich material as their degradation gives rise to high levels of ammonia, which inhibits many microorganisms. A common problem arising in the high-ammonia biogas process is the accumulation of volatile fatty acids, such as acetate, propionate or butyrate, which negatively affects the methane production. The aim of this study was to isolate and identify the syntrophic butyrate-oxidizing bacteria present in the enriched syntrophic communities originating from thermophilic continuously fed laboratory-scale reactors. Butyrate was added to batch assays containing the enrichment cultures. Sequencing of a 464 bp region within the 16S rRNA gene was conducted to study the change in microbial community structure over time during the butyrate degradation. The enrichment culture was also used as an inoculum source during the isolation attempts using agar cultivation, colony transfer and 16S rRNA gene sequencing of the obtained isolates. From the Illumina sequencing data, it could be concluded that the novel species of interest belonged to the genus Syntrophothermus. Two species were isolated, however neither appeared to be the butyrate-degrading bacterium. One of the species was Defluviitoga tunisiensis and the other was a novel species related to the mesophilic bacterium Schnuerera ultunensis.

Les procédés anaérobies de traitement de déchets apparaissent clairement pouvoir répondre à l'enjeu socio-économique actuel que représente la valorisation énergétique de la fraction organique contenue dans les déchets ménagers. En effet, les processus de dégradation anaérobies font intervenir en cascade, différentes réactions et populations de micro-organismes permettant de transformer la matière organique en biogaz riche en méthane. Une bonne connaissance des effets des paramètres opérationnels sur l'orientation des métabolismes s'avère ainsi nécessaire à l'émergence de solutions permettant d'optimiser ces procédés. Ceci est notamment le cas pour la dernière étape, appelée méthanogenèse. Dans ce contexte, l'approche isotopique reposant sur la mesure de la composition isotopique (13C/12C) du méthane et du dioxyde de carbone, devrait pouvoir répondre à cet objectif en permettant l'identification des métabolismes à l'origine de la production du méthane. La transposabilité à l'étude de la digestion anaérobie des déchets de cette approche isotopique déjà utilisée dans les écosystèmes naturels, a tout d'abord été vérifiée expérimentalement. Les effets de certains paramètres opérationnels connus pour avoir un impact fort sur le processus de digestion anaérobie, tels que la température et la concentration en azote ammoniacal, ont ensuite été étudiés. Il a été mis en évidence qu'en condition thermophile, la méthanogenèse acétoclaste observée en condition mésophile, était remplacée par une oxydation syntrophique de l'acétate lors de la digestion anaérobie des déchets ménagers. Des expériences sur acétate ont montré que cet effet sur les voies métaboliques n'était toutefois pas systématique et pourrait ne pas être dû à un effet direct d'une augmentation de la température, mais plutôt à l'accroissement de la concentration en ammoniaque qui en résulte. D'autres expériences ont clairement établi qu'une augmentation de la concentration en azote ammoniacal conduisait également à la mise en place de l'oxydation syntrophique de l'acétate. Le couplage de l'approche isotopique avec des analyses microbiologiques a révélé que cette réaction d'oxydation syntrophique de l'acétate, à haute concentration en azote ammoniacal, pouvait s'établir telle que déjà décrite, par la mise en place d'une relation symbiotique bactéries/archées hydrogénotrophes strictes, mais également de manière différente en impliquant des membres de la famille Methanosarcinaceae qui pourraient réaliser seuls les deux étapes de la réaction (oxydation et méthanogenèse hydrogénotrophe). L'application de l'approche isotopique a également permis de mettre en évidence, lors d'une expérience visant à simuler la recirculation de différents effluents au sein d'une installation de stockage de déchets bioactive, l'influence de la nature de l'effluent sur l'orientation des métabolismes méthanogènes. Enfin, l'influence de la proportion de déchets verts, lors de la co-digestion biodéchets / déchets verts, sur la concentration en ions ammonium libérés ainsi que sur l'orientation du métabolisme en résultant, a été étudiée. Les potentialités d'une utilisation de l'approche isotopique sur site ont également été investiguées au travers d'une campagne de mesures sur une installation de stockage de déchets non dangereux. / Anaerobic waste treatment processes are clearly part of the answer to a current important socio-economic issue in waste management: energy production from the organic fraction of municipal solid waste. The anaerobic digestion of municipal solid waste is a complex process involving numerous reactions and microorganism communities. At the end of the degradation process, some biogas with a particularly high methane content is produced. A detailed knowledge on how operational parameters affect metabolism orientations is required to optimize these treatment processes. This is in particular the case for the last degradation reaction called methanogenesis. In this context, an isotopic approach based on isotopic composition measurements (13C/12C) for methane and carbon dioxide can provide some clues with regard to this objective. Indeed, this methodology enables the determination of the methanogenic pathways by which methane is produced.Transferability of the isotopic approach used for natural ecosystems to the field of anaerobic digestion of municipal solid waste was first experimentally verified. In a second time, the effects of some operational parameters known to strongly impact the anaerobic digestion process, such as temperature and ammonia concentration, were studied. During anaerobic digestion of reconstituted municipal solid waste in thermophilic conditions, it was shown that aceticlastic methanogenesis (occurring in mesophilic conditions) was replaced by a syntrophic acetate oxidation reaction. Additional experiments using acetate as sole substrate were performed and showed that this effect on the metabolic pathways was not systematic. Consequently, it cannot be due to a direct effect of the temperature increase. It could rather be explained by the induced and indirect increase in ammonia concentration. Additional experiments clearly demonstrated that an increase in ammonia concentration led to the establishment of a syntrophic acetate oxidation reaction. The isotopic approach was combined with microbiological analyses and showed that the syntrophic acetate oxidation reaction occurring at high ammonia concentration during acetate incubations could have been performed through a syntrophic relationship between bacteria and strict hydrogenotrophic archaea, as previously described in the literature. Interestingly, the syntrophic acetate oxidation could also have occurred using a different pathway relying on members of the Methanosarcinaceae family putatively able to perform the two steps of the reaction (oxidation and hydrogenotrophic methanogenesis). In addition, the implementation of the isotopic approach during an experiment designed to simulate a landfill bioreactor evidenced the influence of the effluent's nature on the methanogenesis metabolism orientation. The influence of green waste proportion during the co-digestion of biowaste / green waste mixtures on resulting ammonia concentrations and methanogenesis pathways was also studied through dedicated experiments. Finally, the potential of the isotopic approach for landfill-scale application was investigated through a measurement campaign on a landfill site.

<p>Biogas är benämningen för metangas (CH₄) som är producerad via anaerob (syrefri) rötning av biologiskt material. I Linköping finns en av Sveriges största biogasanläggningar. Anläggningen drivs av Svensk Biogas AB som ägs av Tekniska Verken i Linköping AB (publ.). I anläggningen rötas stora mängder proteinrikt substrat vilket leder till höga halter av ammoniak (NH₃) och ammonium (NH₄⁺) i rötkammaren. Ammoniak (NH₃) är toxiskt för de metanbildare som i en välmående process står för den största delen av metangasproduktionen. Höga halter av ammoniak (NH₃) och ammonium (NH₄⁺) kan inhibera dessa metanbildare vilket leder till minskad gasproduktion.</p><p>Detta examensarbete syftar främst till att genom ett kontinuerligt rötkammarexperiment utreda om zeoliter är ett lämpligt hjälpmedel för att reducera ammonium (NH₄⁺) i en anaerob process. Vid sänkta halter ammonium (NH₄⁺) är hypotesen att de mikroorganismer som är aktiva i den mest effektiva metanbildningsvägen återetableras. Arbetet syftar även till att experimentellt utreda vilka effekter zeoliter i sin helhet har på den anaeroba processen. Zeoliters effekt vid låga zeolitkoncentrationer utreds i en serie utrötningsexperiment i batch. Dessutom har en materialstudie kring zeoliternas kapacitet i olika miljöer genomförts.</p><p>Materialstudien visade att den valda zeoliten som studerades, clinoptilolite, hade en maximal katjonbytarkapacitet på ≈ 19 mg NH₄⁺/g zeolit. Vidare konstaterades att zeoliter med mindre diameter än 1 mm har avsevärt bättre kapacitet än zeoliter med större diameter. I det kontinuerliga rötkammarexperimentet konstaterades att clinoptilolite kan användas i en rötkammare för att reducera ammoniumhalten (NH₄⁺). Detta utan att några allvarliga processtörningar uppstår. Cirka 175 g zeolit/l krävdes för att reducera ammoniumhalten (NH₄⁺) från 5300 mg NH₄⁺/l till 3200 mg NH₄⁺/l. Det är inte realistisktatt använda så stora mängder zeoliter i en fullskalig anläggning. Mikrobiologiskt sett observerades ingen förändring av de metanbildarna som dominerar den effektivaste metanbildningsvägen.</p><p>Resultaten från utrötningsförsöket i batch visade att zeolittillsatser av 5 g/l respektive 10 g/l hade en klart positiv effekt på metanbildningen jämfört utan zeolittillsats. I de batcher med 1-10 g zeolit/l startade metangasproduktionen ≈ 14 dagar tidigare än batcherna med 0 g zeolit/l. 16 dagar efter att experimentet startade hade batcherna med 5-10 g zeolit/l producerat ≈ 500 ml metangas (CH₄) jämfört med serien utan zeoliter som vid samma tidpunkt producerat ≈ 75 ml metangas (CH₄). Utrötningsgraden ökade i samtliga serier med zeolittillsats jämfört med serien utan zeoliter. Tillsatsen av 5 g zeolit/l ökade den specifika metangasproduktionen med ≈ 19 % jämfört med utan zeolittillsatser. Slutsatsen är att clinoptilolite i små koncentrationer, mellan 5-10 g/l, påverkar så väl kinetiken som utrötningsgraden för den anaeroba processen på ett positivt sett. Den optimala koncentrationen av clinoptilolite i en mesofil anaerob process bör ligga mellan 5-10 g zeolit/l.</p> / <p>Methane (CH₄) is formed by anaerobic (oxygen-free) digestion of biological materials. One of Sweden's largest biogas plants is placed in Linköping. The plant is operated by Svensk Biogas AB, which is owned by Tekniska Verken i Linköping AB (publ.). In their biogas plant a large amount of protein rich material is handled. High amounts of protein leads to high levels of ammonia (NH₃) and ammonium (NH₄⁺) in the digestion chamber. High levels of ammonia (NH₃) are toxic to the most dominant methane forming microorganism. High concentrations of ammonia (NH₃) and ammonium (NH₄⁺) can inhibit these methane forming microorganisms which may lead to a reduction in gas production.</p><p>This aim with this master thesis was to reduce high ammonium levels by adding zeolites to a lab scale continuous digestion chamber. The hypothesis is that at reduced levels of ammonium (NH₄⁺) the most effective methane forming microorganism will reestablish. This thesis also aims to experimentally investigate all types of effects that zeolites may have in an anaerobic digestion process. Which effect zeolites at low concentration have in a digestion chamber will be investigated by using lab scale batch digestion chambers. In addition, a material study on the capacity of the zeolites in different environments will be investigated.</p><p>The material study showed that the selected zeolite, clinoptilolite, had a cat ion exchange capacity around 19 mg NH₄⁺/g zeolite. It was also found that the zeolites with a diameter less than 1 mm had significantly better capacity than zeolites with larger diameter. In the continuous digestion experiment it was found that clinoptilolite can be used in a digestion chamber to reduce high levels of ammonium (NH₄⁺). This without any serious disorder on the process. Around 175 g zeolite/l was needed to reduce ammonium levels (NH₄⁺) from 5300 mg NH₄⁺/l to 3200 mg NH₄⁺/l. However, it is not realistic to use such large amounts of zeolites in a full-scale digestion chamber. No changes in the culture of methane forming microorganisms were found.</p><p>The results of the batch experiment showed that concentrations of 5 g zeolite/l and 10 g zeolite/l had a positive effect on the methanogenesis compared to batches without additives. In the batches with 1-10 g zeolite/l the forming of methane began about 14 days earlier than in the batches without any zeolites. After 16 days, batches with 5-10 g zeolite/l had produced about 500 ml of methane (CH₄), compared with series without additives, which at the same time had produced about 75 ml of methane (CH₄). The methane yield increased in all series which included zeolites compared to the batches without zeolites. Addition of 5 g zeolite/l increased the specific methane production by approximately 19 % compared to no additives. The conclusion is that clinoptilolite in small concentrations; 5-10 g/l have a positive effect on as well the kinetics as on the methane yield for the anaerobic process. The best concentration of zeolites in a mesophile anaerobic digestion chamber appears to be between 5-10 g zeolite/l.</p>

Biogas är benämningen för metangas (CH4) som är producerad via anaerob (syrefri) rötning av biologiskt material. I Linköping finns en av Sveriges största biogasanläggningar. Anläggningen drivs av Svensk Biogas AB som ägs av Tekniska Verken i Linköping AB (publ.). I anläggningen rötas stora mängder proteinrikt substrat vilket leder till höga halter av ammoniak (NH3) och ammonium (NH4+) i rötkammaren. Ammoniak (NH3) är toxiskt för de metanbildare som i en välmående process står för den största delen av metangasproduktionen. Höga halter av ammoniak (NH3) och ammonium (NH4+) kan inhibera dessa metanbildare vilket leder till minskad gasproduktion. Detta examensarbete syftar främst till att genom ett kontinuerligt rötkammarexperiment utreda om zeoliter är ett lämpligt hjälpmedel för att reducera ammonium (NH4+) i en anaerob process. Vid sänkta halter ammonium (NH4+) är hypotesen att de mikroorganismer som är aktiva i den mest effektiva metanbildningsvägen återetableras. Arbetet syftar även till att experimentellt utreda vilka effekter zeoliter i sin helhet har på den anaeroba processen. Zeoliters effekt vid låga zeolitkoncentrationer utreds i en serie utrötningsexperiment i batch. Dessutom har en materialstudie kring zeoliternas kapacitet i olika miljöer genomförts. Materialstudien visade att den valda zeoliten som studerades, clinoptilolite, hade en maximal katjonbytarkapacitet på ≈ 19 mg NH4+/g zeolit. Vidare konstaterades att zeoliter med mindre diameter än 1 mm har avsevärt bättre kapacitet än zeoliter med större diameter. I det kontinuerliga rötkammarexperimentet konstaterades att clinoptilolite kan användas i en rötkammare för att reducera ammoniumhalten (NH4+). Detta utan att några allvarliga processtörningar uppstår. Cirka 175 g zeolit/l krävdes för att reducera ammoniumhalten (NH4+) från 5300 mg NH4+/l till 3200 mg NH4+/l. Det är inte realistisktatt använda så stora mängder zeoliter i en fullskalig anläggning. Mikrobiologiskt sett observerades ingen förändring av de metanbildarna som dominerar den effektivaste metanbildningsvägen. Resultaten från utrötningsförsöket i batch visade att zeolittillsatser av 5 g/l respektive 10 g/l hade en klart positiv effekt på metanbildningen jämfört utan zeolittillsats. I de batcher med 1-10 g zeolit/l startade metangasproduktionen ≈ 14 dagar tidigare än batcherna med 0 g zeolit/l. 16 dagar efter att experimentet startade hade batcherna med 5-10 g zeolit/l producerat ≈ 500 ml metangas (CH4) jämfört med serien utan zeoliter som vid samma tidpunkt producerat ≈ 75 ml metangas (CH4). Utrötningsgraden ökade i samtliga serier med zeolittillsats jämfört med serien utan zeoliter. Tillsatsen av 5 g zeolit/l ökade den specifika metangasproduktionen med ≈ 19 % jämfört med utan zeolittillsatser. Slutsatsen är att clinoptilolite i små koncentrationer, mellan 5-10 g/l, påverkar så väl kinetiken som utrötningsgraden för den anaeroba processen på ett positivt sett. Den optimala koncentrationen av clinoptilolite i en mesofil anaerob process bör ligga mellan 5-10 g zeolit/l. / Methane (CH4) is formed by anaerobic (oxygen-free) digestion of biological materials. One of Sweden's largest biogas plants is placed in Linköping. The plant is operated by Svensk Biogas AB, which is owned by Tekniska Verken i Linköping AB (publ.). In their biogas plant a large amount of protein rich material is handled. High amounts of protein leads to high levels of ammonia (NH3) and ammonium (NH4+) in the digestion chamber. High levels of ammonia (NH3) are toxic to the most dominant methane forming microorganism. High concentrations of ammonia (NH3) and ammonium (NH4+) can inhibit these methane forming microorganisms which may lead to a reduction in gas production. This aim with this master thesis was to reduce high ammonium levels by adding zeolites to a lab scale continuous digestion chamber. The hypothesis is that at reduced levels of ammonium (NH4+) the most effective methane forming microorganism will reestablish. This thesis also aims to experimentally investigate all types of effects that zeolites may have in an anaerobic digestion process. Which effect zeolites at low concentration have in a digestion chamber will be investigated by using lab scale batch digestion chambers. In addition, a material study on the capacity of the zeolites in different environments will be investigated. The material study showed that the selected zeolite, clinoptilolite, had a cat ion exchange capacity around 19 mg NH4+/g zeolite. It was also found that the zeolites with a diameter less than 1 mm had significantly better capacity than zeolites with larger diameter. In the continuous digestion experiment it was found that clinoptilolite can be used in a digestion chamber to reduce high levels of ammonium (NH4+). This without any serious disorder on the process. Around 175 g zeolite/l was needed to reduce ammonium levels (NH4+) from 5300 mg NH4+/l to 3200 mg NH4+/l. However, it is not realistic to use such large amounts of zeolites in a full-scale digestion chamber. No changes in the culture of methane forming microorganisms were found. The results of the batch experiment showed that concentrations of 5 g zeolite/l and 10 g zeolite/l had a positive effect on the methanogenesis compared to batches without additives. In the batches with 1-10 g zeolite/l the forming of methane began about 14 days earlier than in the batches without any zeolites. After 16 days, batches with 5-10 g zeolite/l had produced about 500 ml of methane (CH4), compared with series without additives, which at the same time had produced about 75 ml of methane (CH4). The methane yield increased in all series which included zeolites compared to the batches without zeolites. Addition of 5 g zeolite/l increased the specific methane production by approximately 19 % compared to no additives. The conclusion is that clinoptilolite in small concentrations; 5-10 g/l have a positive effect on as well the kinetics as on the methane yield for the anaerobic process. The best concentration of zeolites in a mesophile anaerobic digestion chamber appears to be between 5-10 g zeolite/l.