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Anaerobic digestion (AD) is a widespread technology for treating waste streams such as livestock manure. During AD, biogas is produced and subsequently used as renewable energy for certain purposes, such as injection into the natural gas grid or as fuel for transportation. Despite its many advantages, AD can be limited by various factors, including process instability against volatile fatty acids (VFA), nitrogen overloads, or the presence of inhibitors, as well as the need for biogas post-processing to increase its methane content. Therefore, strategies are needed to monitor the AD process, control the effluent quality and upgrade the biogas recovered. Microbial electrochemical technologies (MET) have the potential to optimize AD. MET are systems in which oxidation and/or reduction reactions are catalyzed by electroactive microorganisms (EAM) on the surface of an electrode. Typically, EAM used in AD-MET combinations are dominated by Geobacter spp., that form multilayer biofilms on electrodes (e.g., anodes) used as solid terminal electron acceptors. However, using Geobacter spp. dominated biofilm anodes in AD-MET combinations has so far encountered several hurdles, ranging from biofilm dispersal to inhibition of biofilm performance. Thus, the intention of the present thesis was to identify and to address the different inhibition processes of Geobacter spp. dominated biofilm anodes in AD-MET combinations. Particular attention was devoted to the impact that planktonic methanogens, particles and dissolved components present in AD effluents may have on the activity, stability and microbial community of Geobacter spp. dominated biofilm anodes. This was achieved by investigating the effect of biofilm age, applied anode potentials as well as the role played by specific methanogens with different metabolisms on the activity, stability and microbial community of Geobacter spp. dominated biofilms. The results indicated that older Geobacter spp. dominated biofilm anodes (≥ 5-week-old) are far more active and stable than younger biofilms (≤ 3-week-old) in AD environments. Compared to high applied anode potential (0.4 V vs. Ag/AgCl sat. KCl), low applied anode potentials (-0.2 V to 0.2 V vs. Ag/AgCl sat. KCl) resulted in higher activities of Geobacter spp. dominated biofilm anodes in AD environments. Other results indicated that AD effluents dominated by strict acetoclastic methanogens (e.g., Methanothrix spp.) cause deterioration in biofilm stability and activity, in contrast to AD effluents dominated by hydrogenotrophic methanogens (e.g., Methanobacterium spp.). In conclusion, the thesis provides useful information for understanding and improving the performance of AD-MET combinations and ways to overcome the multiple hurdles encountered so far.:1 Introduction 12 1.1 Basics of microbiology of anaerobic digestion 12 1.1.1 Methanogens and their functionality 12 1.1.2 Anaerobic digestion - methanogens and bacteria 13 1.1.3 Microbial diversity in anaerobic digestion: focus on methanogens 14 1.1.4 Metabolism and syntrophy in anaerobic digestion 16 1.1.5 Parameters influencing anaerobic digestion 18 1.2 Microbial electrochemical technologies 19 1.2.1 Primary microbial electrochemical technologies - possible applications 19 1.2.2 Basic examples of primary microbial electrochemical technologies 21 1.2.3 Geobacter spp. as model electroactive microorganism in microbial electrochemical technologies 23 1.2.4 Modes of electron transfer between electroactive microorganisms and electrodes 24 1.2.5 Electrochemical characterization of biofilm anodes 27 1.3 Combination of anaerobic digestion and microbial electrochemical technologies 28 1.3.1 Benefits: biotechnological applications 28 1.3.2 Challenges: factors limiting the combination anaerobic digestion - microbial electrochemical technologies 29 1.4 Aims and thesis outline 30 2 Publication 1: Benefits of Age – Improved Resistance of Mature Electroactive Biofilm Anodes in Anaerobic Digestion 33 2.1 Supplementary information for “Benefits of Age – Improved Resistance of Mature Electroactive Biofilm Anodes in Anaerobic Digestion” 43 3 Publication 2: Combining Geobacter spp. dominated biofilms and anaerobic digestion effluents - the effect of effluent composition and electrode potential on biofilm activity and stability 55 3.1 Supplementary information for “Combining Geobacter spp. dominated biofilms and anaerobic digestion effluents - the effect of effluent composition and electrode potential on biofilm activity and stability” 67 4 Publication 3: A unified and simple medium for growing model methanogens 91 4.1 Supplementary information for “A unified and simple medium for growing model methanogens” 105 5 Publication 4: Effect of model methanogens on the activity, stability, and microbial community structure of Geobacter spp. dominated biofilm anodes 117 5.1 Supplementary information for “Effect of model methanogens on the activity, stability, and microbial community structure of Geobacter spp. dominated biofilm anodes” 153 6 Discussion 167 6.1 The older the biofilm, the higher its activity and resistance when combined with anaerobic digestion effluents 167 6.2 Low applied anode potential leads to high activity of Geobacter spp. dominated biofilm in anaerobic digestion environments. 168 6.3 The role of methanogens, abiotic particles, dissolved components in the combination anaerobic digestion – microbial electrochemical technologies 170 6.3.1 Abiotic particles, dissolved components present in anaerobic digestion effluents do not always interfere with the activity, stability and community of Geobacter spp. dominated biofilm anodes 170 6.3.2 The activity and community of Geobacter spp. dominated biofilm anodes in anaerobic digestion environments vary with the predominant group of methanogens 171 7 Conclusions and future prospects 173 8 References 176 9 Appendix 187 9.1 Author contribution statements of published articles 187 9.2 Curriculum vitae 193 9.3 List of publications and conference contributions 196 9.4 Acknowledgment 199 9.5 Declaration of authorship 201

Le présent travail s’intéresse à l’étude de la diversité des communautés procaryotiques, basée sur le gène codant pour l’ARNr 16S et sur les gènes codants pour la sous-unité β du sulfite réductase dissimilatrice (dsrB) et la sous-unité alpha de la méthyl-coenzyme M réductase (mcrA), pour étudier la diversité de la communauté des bactéries sulfato-réductrices et des méthanogènes, respectivement à partir des échantillons collectés en saison sèche ou pluvieuse du Chott El Jerid. Les analyses des séquences du gène codant pour l’ARNr 16S ont montré que les bactéries regroupées aux Proteobacteria et Firmicutes sont détectés dans les deux saisons alors que les séquences appartenant au groupe taxonomique Bacteroidetes, Actinobacteria et Betaproteobacteria sont apparues uniquement dans la saison pluvieuse. Le groupe Deinococcus-Thermus sont observés que dans la saison sèche. Dans le domaine des archées, la plupart des séquences appartiennent au phylum Euryachaeota, détecté dans les deux saisons, alors que, le phylum Crenarchaeota apparait uniquement dans la saison pluvieuse. En plus, les bactéries sulfato-réductrices, appartenant à la classe Deltaproteobacteria, sont fréquents notamment à la saison pluvieuse prouvée déjà par les deux techniques DGGE et qPCR. A partir des cultures d’enrichissement, de nombreuses bactéries anaérobies fermentaires appartiennent aux familles Halanaerobiaceae et Halobacteroidaceae. Les analyses phylogénétiques ainsi que les caractéristiques phénotypiques et physiologiques montrent une nouvelle souche Sporohalobacter salinus proche de l’espèce Sporohalobacter lortetii, seule espèce décrite à ce jour du genre Sporohalobacter. / The present work concerns microbial biodiversity of prokaryotic communities, sulfate-reducing bacteria, and methanogens targeting the 16S rRNA gene and functional gene markers encoding the dissimilatory sulfite reductase β-subunit gene (dsrB) and alpha subunit of the methyl-coenzyme M reductase (mcrA), respectively from samples collected in the dry and wet seasons from Chott El Jerid. Phylogenetic analysis targeting the 16S rRNA gene showed that bacteria were grouped to Proteobacteria and Firmicutes detected at both seasons, whereas, Bacteroidetes, Actinobacteria and Betaproteobacteria were present only in the wet season. Deinococcus-Thermus group were observed in the dry season. Archaeal sequences were belonged to the phyla of Euryarchaeota in both seasons and Crenarchaeota was appeared in wet season. Sulfate-reducing bacteria, related to Deltaproteobacteria class were dominant mainly in wet season proved by two techniques DGGE and QPCR. From enrichment cultures, anaerobic fermentative bacteria were isolated in pure cultures, related to Halanaerobiaceae and Halobacteroidaceae families. Phylogenetic analysis, phenotypic and physiological characteristics showed a novel strain Sporohalobacter salinus related to Sporohalobacter lortetii, an unique species of genus Sporohalobacter described until now.

Deep subsurface rock samples from Upper Ordovician strata in the Michigan Basin were analyzed for the presence of microbial communities. High concentrations of biogenic methane were observed in the Upper and Middle Ordovician formations. Total porosity values for the shale, shale hard bed and limestone samples were 7.4%, 2.5% and 1.9%, respectively. Hydrocarbon presence ranged from petroliferous shale, to bituminous layering in shale hard beds, to hydrocarbon odour in limestone. Organic carbon content ranged from 0.5 to 2.5%, highest amount being present in the shale. Environmental DNA was extracted from core samples and PCR amplified using 16S rDNA bacterial primers. PCR performed with archaeal 16S rDNA and methanogen-specific (mcrA) primers did not yield DNA amplification. Gene analysis indicated that bacterial sequences similar to Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were present. Most sequences were not related to known cultivated species. Proteobacteria was the most dominant phyla at all depths and included heterotrophic, lithotrophic, acidophilic, radiotolerant, and sulphate-reducing species of bacteria. This study concludes that the observed biogenic methane is a product of ancient methanogenesis.

Deep subsurface rock samples from Upper Ordovician strata in the Michigan Basin were analyzed for the presence of microbial communities. High concentrations of biogenic methane were observed in the Upper and Middle Ordovician formations. Total porosity values for the shale, shale hard bed and limestone samples were 7.4%, 2.5% and 1.9%, respectively. Hydrocarbon presence ranged from petroliferous shale, to bituminous layering in shale hard beds, to hydrocarbon odour in limestone. Organic carbon content ranged from 0.5 to 2.5%, highest amount being present in the shale. Environmental DNA was extracted from core samples and PCR amplified using 16S rDNA bacterial primers. PCR performed with archaeal 16S rDNA and methanogen-specific (mcrA) primers did not yield DNA amplification. Gene analysis indicated that bacterial sequences similar to Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were present. Most sequences were not related to known cultivated species. Proteobacteria was the most dominant phyla at all depths and included heterotrophic, lithotrophic, acidophilic, radiotolerant, and sulphate-reducing species of bacteria. This study concludes that the observed biogenic methane is a product of ancient methanogenesis.

Deep subsurface rock samples from Upper Ordovician strata in the Michigan Basin were analyzed for the presence of microbial communities. High concentrations of biogenic methane were observed in the Upper and Middle Ordovician formations. Total porosity values for the shale, shale hard bed and limestone samples were 7.4%, 2.5% and 1.9%, respectively. Hydrocarbon presence ranged from petroliferous shale, to bituminous layering in shale hard beds, to hydrocarbon odour in limestone. Organic carbon content ranged from 0.5 to 2.5%, highest amount being present in the shale. Environmental DNA was extracted from core samples and PCR amplified using 16S rDNA bacterial primers. PCR performed with archaeal 16S rDNA and methanogen-specific (mcrA) primers did not yield DNA amplification. Gene analysis indicated that bacterial sequences similar to Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were present. Most sequences were not related to known cultivated species. Proteobacteria was the most dominant phyla at all depths and included heterotrophic, lithotrophic, acidophilic, radiotolerant, and sulphate-reducing species of bacteria. This study concludes that the observed biogenic methane is a product of ancient methanogenesis.

Deep subsurface rock samples from Upper Ordovician strata in the Michigan Basin were analyzed for the presence of microbial communities. High concentrations of biogenic methane were observed in the Upper and Middle Ordovician formations. Total porosity values for the shale, shale hard bed and limestone samples were 7.4%, 2.5% and 1.9%, respectively. Hydrocarbon presence ranged from petroliferous shale, to bituminous layering in shale hard beds, to hydrocarbon odour in limestone. Organic carbon content ranged from 0.5 to 2.5%, highest amount being present in the shale. Environmental DNA was extracted from core samples and PCR amplified using 16S rDNA bacterial primers. PCR performed with archaeal 16S rDNA and methanogen-specific (mcrA) primers did not yield DNA amplification. Gene analysis indicated that bacterial sequences similar to Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were present. Most sequences were not related to known cultivated species. Proteobacteria was the most dominant phyla at all depths and included heterotrophic, lithotrophic, acidophilic, radiotolerant, and sulphate-reducing species of bacteria. This study concludes that the observed biogenic methane is a product of ancient methanogenesis.

Myrmarker har en stor roll i regleringen av den globala kolbalansen och koncentrationerna av koldioxid och metan i atmosfären, vilket gör dem till speciellt viktiga ekosystem ur ett klimatförandringsperspektiv. Förändringar av myrmarker genom naturlig utveckling eller antropogen påverkan kan därför få långtgående störningar av myrars klimatreglerande funktion. Mikroorganismer har en avgörande roll i biogeokemiska processer genom att t ex bryta ned organisk material i mark och därmed styra kolets kretslopp. För att förstå hur myrsystemen reagerar på störningar är det därför väsentligt att veta hur mikroorganismsamhällena reagerar genom förändringar i sammansättning och biogeokemisk aktivitet. Målet för studierna, som ligger till grund för denna avhandling, var att undersöka hur mikroorganismsamhällen i myrar reagerar på uppvärmning genom klimatförändring och ökade kväve- (N) och svavel- (S) halter i nederbörd. High through-put sekvensering användes för att studera taxonomiska och funktionella egenskaper hos mikroorganismerna i myrar och quantative PCR användes för att mer specifikt studera de metanbildande arkeorna. Två fältkampanjer vardera omfattande tre ombrotrofa myrar med olika klimatförhållanden och olika mängder N och S inederbörden användes för att undersöka lokala och storskaliga effekter på myrars mikrobiella samhällen. Resultaten visade att latudinell variation i geoklimatologiska förhållanden (temperatur ochnederbördsmängd) och deposition av näringsämnen hade en påverkan på sammansättningen av de mikrobiella samhällena och aktiva metanbildare förr än variationen i den kemiska miljön inom varje specifik myr. Myrväxtsamhällenas sammansättning för en specifik myr visades sig i stor utsträckning styra sammansättningen av motsvarande mikrobiella samhälle i torvprofilen. Detta framgick klart av i en analys av samexisterande nätverk av mikroorganismsamhällen och motsvarande växtsamhällen i en studie av tre geografiskt skilda myrar med olika kvävedeposition. Effekterna av klimatförändring och nederbörd med olika mängder av N och S studerades mer specifikt genom att analysera de mikrobiellasamhällena i  ett långliggande (18 år) försök. Påverkan av var och en av dessa manipulationer antingen förstärktes eller minskades, när de förekom i kombinationer. Ökad kvävedeposition var den faktor som hade starkast effekt. De långvariga störningarna medförde stora förändringar i den mikrobiella taxonomin inom samhällena. Detta återspeglades dock inte i den fysiologiska kapaciteten, vilket visar att det finns en stark buffring i myrarnas mikrobiella funktion. Detta tyder på att framtida utveckling av myrar i relation till olika störningar sannolikt inte kommer att påverka myrarnas roll för kolbalans och växthusgasutbyte med atmosfären. / Peatlands play a substantial role in regulating the global carbon balance and concentrations of the greenhouse gases CO2 and CH4 in the atmosphere, and are thus of utmost importance from a climate change perspective. Any changes of peatland functions due to natural or anthropogenic perturbations may result in changes in these ecosystem services. Soil microbial communities are essential drivers of biogeochemical processes, including the carbon cycle. In order to fully understand the effect of environmental perturbations on peatland functions, it is essential to understand how microbial communities are affected. The aim of the research presented in this thesis was to investigate the responses of the peat microbial communities to climate change and increased precipitation of nitrogen(N) and sulfur (S) compounds. High-throughput sequencing approaches were used to investigate the taxonomic and functional composition of microbial communities, and quantitative PCR was used to specifically target the methanogen community. Two field studies including three ombrotrophic peatlands each that differed in climatological conditions and atmospheric N and S depositions, were used to investigate and compare the effect of large- and local-scale environmental conditions on microbial communities. The results show that the variation in geo-climatological (temperature and precipitation) and atmospheric deposition conditions along the latitudinal gradient modulate the peat microbial community composition and the abundance of active methanogens to a greater extent thansite-related microhabitats. Furthermore, a tight coupling between the plant community composition of a site and the composition of its microbial community was observed, and was found to be mainly driven by plants rather than microorganisms. These co-occurrence networks are strongly affected by seasonal climate variability and the interactions between species in colder areas are more sensitive to climate change. The long-term effects of warming and increased N and S depositions on the peat microbial communities were further investigated using an 18-year in-situ peatland experiment simulating these perturbations. The impacts of each of these perturbations on the microbial community were found to either multiply or counteract one another, with enhanced N deposition being the most important factor. While the long-term perturbations resulted in a substantial shift in the taxonomic composition of microbial communities, only minor changes occurred in genome-encoded functional traits, indicating a functional redundancy. This could act as a buffer maintaining ecosystem functioning when challenged by multiple stressors, and could limit future changes in greenhouse gases and carbonexchange.

Au cours des dernières décennies, l’intérêt pour la gazéification de biomasses a considérablement augmenté, notamment en raison de la grande efficacité de recouvrement énergétique de ce procédé par rapport aux autres procédés de génération de bioénergies. Les composants majoritaires du gaz de synthèse, le monoxyde de carbone (CO) et l’hydrogène (H2) peuvent entre autres servir de substrats à divers microorganismes qui peuvent produire une variété de molécules chimiques d’intérêts, ou encore produire des biocarburants, particulièrement le méthane. Il est donc important d'étudier les consortiums méthanogènes naturels qui, en syntrophie, serait en mesure de convertir le gaz de synthèse en carburants utiles. Cette étude évalue principalement le potentiel de méthanisation du CO par un consortium microbien issu d’un réacteur de type UASB, ainsi que les voies métaboliques impliquées dans cette conversion en conditions mésophiles. Des tests d’activité ont donc été réalisés avec la boue anaérobie du réacteur sous différentes pressions partielles de CO variant de 0.1 à 1,65 atm (0.09 à 1.31 mmol CO/L), en présence ou absence de certains inhibiteurs métaboliques spécifiques. Dès le départ, la boue non acclimatée au CO présente une activité carboxidotrophique relativement intéressante et permet une croissance sur le CO. Les tests effectués avec de l’acide 2- bromoethanesulfonique (BES) ou avec de la vancomycine démontrent que le CO est majoritairement consommé par les bactéries acétogènes avant d’être converti en méthane par les méthanogènes acétotrophes. De plus, un plus grand potentiel de méthanisation a pu être atteint sous une atmosphère constituée uniquement de CO en acclimatant auparavant la boue. Cette adaptation est caractérisée par un changement dans la population microbienne désormais dominée par les méthanogènes hydrogénotrophes. Ceci suggère un potentiel de production à large échelle de biométhane à partir du gaz de synthèse avec l’aide de biofilms anaérobies. / Syngas produced through the thermal gasification of biomass for energy recovery has received increased attention in the past decades due to its higher efficiency compared to other bioenergy processes. The gas components of syngas, CO and H2, can serve as substrates for the conversion of desirable chemicals and fuels, namely methane, by a wide range of microorganisms. Meanwhile, anaerobic wastewater-treating sludges have been reported as good sources of carboxidotrophic microorganisms which can be exploited for methane production. Thus it is important to investigate existing methanogenic consortiums which, in syntrophy, are able to convert syngas into useful fuels. This study is mainly focused on the assessment of the carboxidotrophic methanogenic potential present in a natural consortium of microorganisms from a UASB reactor and the identification of CO conversion routes to methane under mesophilic temperatures. To achieve this, a series of kinetic-activity tests with the anaerobic sludge were performed under CO partial pressures varying from 0.1 to 1.65 atm (0.09-1.31 mmol/L) in both the presence and absence of specific metabolic inhibitors. The non-adapted sludge presented an interesting carboxidotrophic activity potential for growing conditions on CO alone. Inhibition experiments with 2- bromoethanesulfonic acid (BES) and vancomycin showed that CO was converted mainly to acetate by acetogenic bacteria, which was further transformed to methane by acetoclastic methanogens. Moreover, it was possible to achieve higher methanogenic potential under 100% CO by acclimation of the sludge. This adaptation led to a shift in the microbial population predominated by hydrogenophilic methanogens. This suggests a possible enrichment potential with anaerobic biofilms for large scale methane production from CO-rich syngas, and further advances the knowledge base for anaerobic reactor development.

[Truncated abstract] In Australia, there is pressure to reduce the amount of methane produced by ruminant livestock because they are the single largest source of methane emitted from anthropogenic sources, accounting for 70.7% of agricultural methane emissions. In addition, methane production represents a loss of gross energy intake to the animal. The organisms that are responsible for methane production in the animal gut are a distinct group of Archaea called methanogens. Methanogens occupy three different niches within the rumen. Some live freely in the rumen digesta (planktonic), others are attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside within the ciliates (endosymbiotic). The types and number of methanogens, as well as rumen ciliates and their symbiotic interactions, influence the amount of methane produced from the rumen. These factors in turn are affected by many factors, including diet and ruminal retention time. In this thesis, I tested the general hypothesis that increasing the amount of grain in the diet and reducing the retention time would affect the abundance and diversity of methanogens in their different niches, including their association with ruminal ciliates. Twenty-four fistulated sheep were used in a complete factorial design with the sheep randomly divided into four groups. ... The change in DGGE banding patterns and Shannon indices when sheep were fed grain indicated that the types of methanogens changed when sheep were fed low and high grain diets, but their diversity did not. In contrast, the diversity of rumen ciliates decreased when sheep were fed a high grain diet. A total of 18 bands from the DGGE analysis of the ciliates were sequenced. All except one, which was 98% similar to Cycloposthium sp. not found previously in the rumen, matched the sequences for previously identified rumen ciliates. Some of the rumen ciliates identified were not present in sheep fed the high grain diet. On a high grain diet, methanogens associate endosymbiotically with rumen ciliates to get better access to hydrogen. It appears that the association between methanogens and rumen ciliates is dictated by the availability of hydrogen in the rumen and not the generic composition of the ciliate population. Furthermore, endosymbiotic methanogens appear to produce less methane than methanogens in other niches. The pot scrubbers did not change ruminal retention time but they did reduce the acetate/propionate measurements observed in sheep on the high grain treatment. The reason why pot scrubbers had this effect remains unknown, but it is interesting to consider that some physical interaction has occurred between the pot scrubbers, the grain and the sheep that has improved the fermentation parameters in sheep fed a high grain diet. The results from this study have advanced our understanding of the interaction between methanogens and ruminal ciliates, and methanogenesis in the rumen in response to dietary changes and mechanical challenges. Extending this work to look more specifically at the species of methanogens that are most closely linked to high methane production and how they interact with the ruminal ciliates will be critical for manipulating enteric greenhouse gas emissions.