Biogas is produced during anaerobic degradation of organic material and mainly consist of methane and carbon dioxide. During the anaerobic degradation, the products are fatty acids such as propionate, butyrate and acetate. In the biogas process a syntrophic cooperation between different microorganisms are important, this involves syntrophic acetate oxidizing bacteria (SAOB) which degrade acetate, syntrophic propionate oxidizing bacteria (SPOB) which degrade propionate and methanogens which produces methane. The propionate degradation is often the slowest step in the biogas process since the microorganism involved do not get much energy from it. It has been observed that FeSO4 and FeCl2 improved the propionate degradation rate in thermophilic SPOB enrichment cultures but no impact on mesophilic enrichment cultures was observed.  The aim of this study was to determine the impact by Fe/S-addition on the key species (SAOB, SPOB and methanogens) involved in the propionate degradation in mesophilic enrichment cultures and cultures with sludge with high ammonia concentration. Furthermore, cultivation studies are performed to reveal the optimal concentrations of iron (FeCl2), which boost the propionate degradation rate in mesophilic SPOB-cultures.  The cultivation study was performed in batches (<500 mL) with 50 mM propionate, 0.3 mM ammonium and different FeCl2 concentrations between 0 to 20 mM. To determine the optimal FeCl2 concentration, the cultures were analysed by measuring the propionate, methane and acetate concentration using High Performance Liquid Chromatography (HPLC). The mesophilic enrichment cultures were treated with FeSO4, FeCl2, NaSO4, FeS, FeCl3, Fe2(SO4)3 and Na2S. The mesophilic cultures with sludge were treated with FeSO4, FeCl2, FeCl3, and a combination of all three compounds. The impact of Fe/S compounds on SAOB, SPOB and methanogens were analysed on the mesophilic enrichment cultures and cultures with sludge by measuring the copies of 16S rRNA gene using quantitative PCR (qPCR). It could be concluded that in the mesophilic enrichment culture none of the Fe/S compounds affected the SPOB or methanogens, but FeS increased the concentration of SAOB. In the mesophilic culture with sludge, FeCl2 and FeCl3 increased the SPOB level, however, FeSO4 decreased the SPOB level. Moreover, the SAOB levels decreased for FeCl3 and when all three compounds were combined. The methanogen level decreased over time by the compounds FeCl2 and FeSO4. From the cultivation study, the optimal FeCl2 concentration was 20 mM.

Anaerobic digestion (AD) is widely used in wastewater treatment plants for stabilisation of primary and waste activated sludges. Increasingly energy prices as well as stringent environmental and public health regulations ensure the ongoing popularity of anaerobic digestion. Reduction of volatile solids, methane production and pathogen reduction are the major objectives of anaerobic digestion. Phased anaerobic digestion is a promising technology that may allow improved volatile solids destruction and methane gas production. In AD models, microbially-mediated processes are described by functionally-grouped microorganisms. Ignoring the presence of functionally-different species in the separate phases may influence the output of AD modeling. The objective of this research was to thoroughly investigate the kinetics of hydrolysis, acetogenesis (i.e., propionate oxidation) and methanogenesis (i.e., acetoclastic) in phased anaerobic digestion systems. Using a denaturing gradient gel electrophoresis (DGGE) technique, bacterial and archaeal communities were compared to complement kinetics studies. Four phased digesters including Mesophilic-Mesophilic, Thermophilic-Mesophilic, Thermophilic-Thermophilic and Mesophilic-Thermophilic were employed to investigate the influence of phase separation and temperature on the microbial activity of the digestion systems. Two more digesters were used as control, one at mesophilic 35 0C (C1) and one at thermophilic 55 0C (C2) temperatures. The HRTs in the first-phase, second-phase and single-phase digesters were approximately 3.5, 14, and 17 days, respectively. All the digesters were fed a mixture of primary and secondary sludges. Following achievement of steady-state in the digesters, a series of batch experiments were conducted off-line to study the impact of the digester conditions on the kinetics of above-mentioned processes. A Monod-type equation was used to study the kinetics of acetoclastic methanogens and POB in the digesters, while a first-order model was used for the investigation of hydrolysis kinetics. Application of an elevated temperature (55 0C) in the first-phase was found to be effective in enhancing solubilisation of particulate organics. This improvement was more significant for nitrogen-containing material (28%) as compared to the PCOD removal (5%) when the M1 and T1 digesters were compared. Among all the configurations, the highest PCOD removal was achieved in the T1T2 system (pvalue<0.05). In contrast to the solubilisation efficiencies, the mesophilic digesters (C1, M1M2 and T1M3) outperformed the thermophilic digesters (C2, T1T2 and M1T3) in COD removal. The highest COD removal was obtained in the T1M3 digestion system, indicating a COD removal efficiency of 50.7±2.1%. The DGGE fingerprints from digesters demonstrated that digester parameters (i.e., phase separation and temperature) influenced the structure of the bacterial and archaeal communities. This resulted in distinct clustering of DGGE profiles from the 1st-phase digesters as compared to the 2nd-phase digesters and from the mesophilic digesters as compared to the thermophilic ones. Based on the bio-kinetic parameters estimated for the various digesters and analysis of the confidence regions of the kinetic sets (kmax and Ks), the batch experiment studies revealed that the kinetic characteristics of the acetoclastic methanogens and POB developed in the heavily loaded digesters (M1 and T1) were different from those species developed in the remaining mesophilic digesters (M2, M3 and C1). As with the results from the mesophilic digesters, a similar observation was made for the thermophilic digesters. The species of acetoclastic methanogens and POB within the T1 digester had greater kmax and Ks values in comparison to the values of the T3 and C2 digesters. However, the bio-kinetic parameters of the T2 digester showed a confidence region that overlapped with both the T1 and T3 digesters. The acetate and propionate concentrations in the digesters supported these results. The acetate and propionate concentrations in the M1 digesters were, respectively, 338±48 and 219±17 mgCOD/L, while those of the M2, M3 and C1 digesters were less than 60 mg/L as COD. The acetate and propionate concentrations were, respectively, 872±38 and 1220±66 in T1 digester, whereas their concentrations ranged 140-184 and 209-309 mg/L as COD in the T2, T3 and C2 digesters. In addition, the DGGE results displayed further evidence on the differing microbial community in the 1st- and 2nd-phase digesters. Two first-order hydrolysis models (single- and dual-pathway) were employed to study the hydrolysis process in the phased and single-stage digesters. The results demonstrated that the dual-pathway hydrolysis model better fit the particulate COD solubilisation as compared to the single-pathway model. The slowly (F0,s) and rapidly (F0,r) hydrolysable fractions of the raw sludge were 36% and 25%, respectively. A comparison of the estimated coefficients for the mesophilic digesters revealed that the hydrolysis coefficients (both Khyd,s and Khyd,r) of the M1 digester were greater than those of the M2 and M3 digesters. In the thermophilic digesters it was observed that the Khyd,r value of the T1 digester differed from those of the T2, T3 and C2 digesters; whereas, the hydrolysis rate of slowly hydrolysable matter (i.e., Khyd,s) did not differ significantly among these digesters. The influence of the facultative bacteria, that originated from the WAS fraction of the raw sludge, and/or the presence of hydrolytic biomass with different enzymatic systems may have contributed to the different hydrolysis rates in the M1 and T1 digesters from the corresponding mesophilic (i.e, M2 and M3) and thermophilic (i.e., T2 and T3) 2nd-phase digesters.

Degradation of the several million tons of solid waste produced in the U.S. annually is microbially mediated, yet little is known about the structure of prokaryotic communities actively involved in the waste degradation process. In the first study, leachates generated during degradation of municipal solid waste (MSW) in the presence (co-disposal) or absence of biosolids were analyzed using laboratory-scale bioreactors over an eight-month period. Archaeal and bacterial community structures were investigated by denaturing gradient gel electrophoresis (DGGE) targeting 16S rRNA genes. Regardless of waste composition, microbial communities in bioreactor leachates exhibited high diversity and temporal trends. Methanogen sequences from a co-disposal bioreactor were predominantly affiliated with the orders Methanosarcinales and Methanomicrobiales. Effect of moisture content on indicator organism (IO) survival during waste degradation was studied using culture-based methods. Fecal coliform and Enterococcus concentrations in leachate decreased below detection limits within fifty days of bioreactor operation during the hydrated phase. IOs could be recovered from the bioreactor leachate even after a prolonged dry period. This study advances the basic understanding of changes in the microbial community during solid waste decomposition. The purpose of the second study was to compare the ability of BOX-PCR to determine genetic relatedness with that of the "gold standard" method, 16S rRNA gene sequencing. BOX-PCR typing could clearly differentiate the strains within different Enterococcus species but closely related genera were not as distinguishable. In contrast, 16S rRNA gene sequencing clearly differentiates between closely related genera but cannot distinguish between different strains of Enterococcus species. This study adds to our knowledge of genetic relationships of enterococci portrayed by two separate molecular methods. The incidence of vancomycin resistant enterococci (VRE) in environmental matrices, residential and hospital wastewater was also investigated. Low-level VRE ( vanC genotype) were isolated from environmental matrices and residential wastewater. VRE isolates from hospital wastewater were identified as E. faecium and demonstrated resistance to ampicillin, ciprofloxacin and vancomycin (vanA genotype), but sensitivity to chloramphenicol and rifampin. Although no high-level VRE were isolated from surface waters, the high proportion of low-level VRE in environmental matrices is a cause for concern from the public health perspective.

Methane is a greenhouse gas and a major contributor to climate change. Methanogenic Archaea produce more than 1 billion tons of this gas each year through methanogenesis, the anaerobic reduction of CO₂ to methane. Coenzyme B (CoB) is one of eight coenzymes required for methanogenesis and it is unique to methanogens. Therefore, this coenzyme is a potential target for inhibiting methanogenesis. To further elucidate the CoB biosynthetic pathway, genes from Methanocaldococcus jannaschii were cloned and expressed in an effort to identify the CoB homoaconitase. From this study, the MJ0499-MJ1277 pair of proteins was identified as the methanogen isopropylmalate isomerase involved in leucine and isoleucine biosynthesis. The MJ1003-MJ1271 pair of proteins was characterized as the homoaconitase required for CoB biosynthesis. This enzyme exhibited broad substrate specificity, catalyzing the isomerization of cis-unsaturated tri-carboxylates with [gamma]-chains of 1-5 methylenes in length. Previously characterized homoaconitases only catalyzed half of the predicted reactions in the isomerization of homocitrate. The MJ1003-MJ1271 proteins function as the first homoaconitase described to catalyze the full isomerization of homocitrate to homoisocitrate. Also, the CoB homoaconitase was identified as specific for (R)-homocitrate and cis-unsaturated intermediates, contrary to a previous study that suggested the substrate specificity of this enzyme included (S)-homocitrate and trans-homoaconitate. The M. jannaschii isopropylmalate isomerase and homoaconitase share more than 50% sequence identity and catalyze analogous reactions. Site directed mutagenesis of the MJ1271 protein was used to identify residues involved in substrate specificity. Arg26 of MJ1271 was critical for the specificity of the CoB homoaconitase. Mutation of this residue to the analogous residue in the M. jannaschii isopropylmalate isomerase, Val28, altered the substrate specificity of the homoaconitase to include the substrates of isopropylmalate isomerase. These homologs of aconitase require a [4Fe-4S] cluster for coordinating their respective substrates at the enzyme active site. However, methanogens lack most of the proteins required for iron-sulfur cluster assembly. Therefore, genes homologous to the Salmonella enterica ApbC iron-sulfur scaffold protein were characterized from methanogens. The MMP0704, MJ0283, and SSO0460 proteins from Methanococcus maripaludis, M. jannaschii, and Solfolobus solfataricus, respectively, were identified as scaffold proteins involved in methanogen iron-sulfur cluster biosynthesis. / text

Anaerobic digestion (AD) is widely used in wastewater treatment plants for stabilisation of primary and waste activated sludges. Increasingly energy prices as well as stringent environmental and public health regulations ensure the ongoing popularity of anaerobic digestion. Reduction of volatile solids, methane production and pathogen reduction are the major objectives of anaerobic digestion. Phased anaerobic digestion is a promising technology that may allow improved volatile solids destruction and methane gas production. In AD models, microbially-mediated processes are described by functionally-grouped microorganisms. Ignoring the presence of functionally-different species in the separate phases may influence the output of AD modeling. The objective of this research was to thoroughly investigate the kinetics of hydrolysis, acetogenesis (i.e., propionate oxidation) and methanogenesis (i.e., acetoclastic) in phased anaerobic digestion systems. Using a denaturing gradient gel electrophoresis (DGGE) technique, bacterial and archaeal communities were compared to complement kinetics studies. Four phased digesters including Mesophilic-Mesophilic, Thermophilic-Mesophilic, Thermophilic-Thermophilic and Mesophilic-Thermophilic were employed to investigate the influence of phase separation and temperature on the microbial activity of the digestion systems. Two more digesters were used as control, one at mesophilic 35 0C (C1) and one at thermophilic 55 0C (C2) temperatures. The HRTs in the first-phase, second-phase and single-phase digesters were approximately 3.5, 14, and 17 days, respectively. All the digesters were fed a mixture of primary and secondary sludges. Following achievement of steady-state in the digesters, a series of batch experiments were conducted off-line to study the impact of the digester conditions on the kinetics of above-mentioned processes. A Monod-type equation was used to study the kinetics of acetoclastic methanogens and POB in the digesters, while a first-order model was used for the investigation of hydrolysis kinetics. Application of an elevated temperature (55 0C) in the first-phase was found to be effective in enhancing solubilisation of particulate organics. This improvement was more significant for nitrogen-containing material (28%) as compared to the PCOD removal (5%) when the M1 and T1 digesters were compared. Among all the configurations, the highest PCOD removal was achieved in the T1T2 system (pvalue<0.05). In contrast to the solubilisation efficiencies, the mesophilic digesters (C1, M1M2 and T1M3) outperformed the thermophilic digesters (C2, T1T2 and M1T3) in COD removal. The highest COD removal was obtained in the T1M3 digestion system, indicating a COD removal efficiency of 50.7±2.1%. The DGGE fingerprints from digesters demonstrated that digester parameters (i.e., phase separation and temperature) influenced the structure of the bacterial and archaeal communities. This resulted in distinct clustering of DGGE profiles from the 1st-phase digesters as compared to the 2nd-phase digesters and from the mesophilic digesters as compared to the thermophilic ones. Based on the bio-kinetic parameters estimated for the various digesters and analysis of the confidence regions of the kinetic sets (kmax and Ks), the batch experiment studies revealed that the kinetic characteristics of the acetoclastic methanogens and POB developed in the heavily loaded digesters (M1 and T1) were different from those species developed in the remaining mesophilic digesters (M2, M3 and C1). As with the results from the mesophilic digesters, a similar observation was made for the thermophilic digesters. The species of acetoclastic methanogens and POB within the T1 digester had greater kmax and Ks values in comparison to the values of the T3 and C2 digesters. However, the bio-kinetic parameters of the T2 digester showed a confidence region that overlapped with both the T1 and T3 digesters. The acetate and propionate concentrations in the digesters supported these results. The acetate and propionate concentrations in the M1 digesters were, respectively, 338±48 and 219±17 mgCOD/L, while those of the M2, M3 and C1 digesters were less than 60 mg/L as COD. The acetate and propionate concentrations were, respectively, 872±38 and 1220±66 in T1 digester, whereas their concentrations ranged 140-184 and 209-309 mg/L as COD in the T2, T3 and C2 digesters. In addition, the DGGE results displayed further evidence on the differing microbial community in the 1st- and 2nd-phase digesters. Two first-order hydrolysis models (single- and dual-pathway) were employed to study the hydrolysis process in the phased and single-stage digesters. The results demonstrated that the dual-pathway hydrolysis model better fit the particulate COD solubilisation as compared to the single-pathway model. The slowly (F0,s) and rapidly (F0,r) hydrolysable fractions of the raw sludge were 36% and 25%, respectively. A comparison of the estimated coefficients for the mesophilic digesters revealed that the hydrolysis coefficients (both Khyd,s and Khyd,r) of the M1 digester were greater than those of the M2 and M3 digesters. In the thermophilic digesters it was observed that the Khyd,r value of the T1 digester differed from those of the T2, T3 and C2 digesters; whereas, the hydrolysis rate of slowly hydrolysable matter (i.e., Khyd,s) did not differ significantly among these digesters. The influence of the facultative bacteria, that originated from the WAS fraction of the raw sludge, and/or the presence of hydrolytic biomass with different enzymatic systems may have contributed to the different hydrolysis rates in the M1 and T1 digesters from the corresponding mesophilic (i.e, M2 and M3) and thermophilic (i.e., T2 and T3) 2nd-phase digesters.

Methane (CH4) plays an important role in regulating Earth’s climate. Its atmospheric concentrations are related to both biotic and abiotic processes. The biotic one can be formed either by chemoautotrophic or heterotrophic pathways by methanogens. Abiotic CH4 formation can occur from several sequential reactions starting with H2 production by serpentinization of Fe-bearing minerals followed by Fischer-Tropsch Type reactions or thermogenic reactions from hydrocarbons. In the presence of suitable electron acceptors, microbial oxidation utilizes CH4 and contributes to regulating its emission.  From the perspectives of astrobiology and Earth climate regulation, this thesis focuses on: (1) Dynamics of CH4 formation and oxidation in lake sediments (Paper I), (2) Constructing an automatic flux chamber to facilitate its emission measurements (Paper II), (3) dynamics of both abiotic and biotic CH4 formation processes related to olivine water interaction in temperature range 30 - 70°C (Paper III and IV). Paper I showed that potential CH4 oxidation strongly correlated to in situ its formation rates across a wide variety of lake sediments. This means that the oxidation rates could be enhanced in environments having the high formation rates. Thereby, the oxidation would likely be able to keep up with potentially increasing the formation rates, as a result diffusive CH4 release from freshwater sediments might not necessarily increase due to global warming. Paper II presented a new automated approach to assess temporal variability of its aquatic fluxes. Paper III and IV together revealed that H2 can be formed via olivine-water interaction. Abiotic CH4 formation was formed likely by Fischer-Tropsch Type reactions at low inorganic carbon concentration but by thermogenic processes at high inorganic carbon concentration. Paper IV showed that biotic methanogenic metabolism could harvest H2 and produce CH4. The dynamics of these processes seemed strongly affected by carbonate chemistry. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Submitted.

Les microorganismes anaérobies représentent la population majoritaire de notre tube digestif et ont un impact remarquable sur notre santé. Leur culture demeure à ce jour longue, fastidieuse et coûteuse et nombreux sont ceux qui restent incultivables. Or la culture est un outil indispensable pour l'étude du microbiote digestif. Ainsi, le laboratoire dans lequel ma thèse s’est déroulée a créé un nouveau concept de culture « Microbial Culturomics » qui a permis d’isoler 193 nouvelles espèces bactériennes anaérobies. Un travail sur l’utilisation des antioxydants pour permettre la culture aérobie des bactéries anaérobies a également été amorcé : une optimisation des techniques de culture prometteuse autour de laquelle mes travaux ont vu le jour. Notre premier projet a consisté à développer un dispositif de culture innovant permettant la culture des archaea méthanogènes en aérobiose et en absence de source externe de dihydrogène. Notre deuxième projet a consisté à élaborer un flacon d’hémoculture unique dans lequel la croissance de toutes les bactéries, aérobies et anaérobies, pouvaient être détectées. Notre troisième projet quant à lui repose sur la comparaison du mode de culture anaérobie et de celui en aérobie avec les antioxydants à travers l’exemple de trois souches bactériennes strictement anaérobies. L’utilisation des antioxydants pour faciliter la culture des microorganismes anaérobies a donc apporter des résultats très prometteurs qui pourrait être utilisés, après validation par des études multicentriques dans les laboratoires de microbiologie clinique et environnementaux. / Anaerobic microorganisms are characterized by their ability to grow and survive in the absence of oxygen. Indeed free oxygen molecules are not used for their metabolism and can be toxic to varying degrees, sometimes leading to cell death. Although it is known that these microorganisms are the predominant in our digestive microbiota and that they have a great impact on our health, their culture remain long, fastidious, costly, and in most cases impossible. Becteria culture is an indispensable tool for isolating strains, performing studies from living models, and identifying new ones. Thus, the laboratory in which my thesis tooks place created a new concept of culture "Microbial Culturomics" which made it possible to isolate 193 new anaerobic bacterial species. A work based on the use of antioxidants to enable the aerobic culture of anaerobic bacteria was also initiated: a promising optimization of the culture techniques from which my work was born. Our first project consisted in developing an innovative culture device allowing the cultivation of methanogenic archaea in aerobic and without an external source of dihydrogen. In our second project, we performed a single culture bottle in which the growth of all bacteria, aerobic and anaerobic, could be detected. Our third project was based on the comparison of anaerobic and aerobic culture with antioxidants through the example of three strictly anaerobic bacterial strains.Therefore the use of antioxidants enable to facilitate anaerobic bacteria cultivation. These results are very encouraging for clinical and environmental microbiology laboratories.

Master of Science / Department of Geology / Matthew Kirk / A growing body of evidence demonstrates that methanogenesis and Fe(III) reduction can occur simultaneously. However, environmental controls on interactions between each are poorly understood. In this study we considered pH as a control on interactions between Fe(III) reduction and methanogenesis in anoxic sediment bioreactors. The reactors consisted of 100mL of synthetic aqueous media, and 1 g of marsh sediment amended with goethite (1mmol). One set of reactors received acidic media (pH 6), and the other alkaline media (pH 7.5). Each set received media containing acetate (0.25 mM) to serve as an electron donor. Control reactors, deficient in acetate, were also included. We maintained a fluid residence time of 35 days by sampling and feeding the reactors every seven days. For pH 6.0 and pH 7.5 reactors, the measured pH of effluent samples averaged 6.33 and 7.37, respectively. The extent of Fe(III) reduction and methanogenesis varied considerably between each set of reactors. More Fe(III) was reduced in the pH 6 reactors (646.39 μmoles on avg.) than the pH 7.5 reactors (31.32 μmoles on avg.). Conversely, more methane formed in pH 7.5 reactors (127.5 μmoles on avg.) than the pH 6 reactors (78.9 μmoles on avg.). Alkalinity concentrations during the middle and end of the experiment averaged 9.6 meq/L and 5.2 meq/L in pH 6 and pH7.5 reactors, respectively Although much less Fe(III) reduction occurred in pH 7.5 reactors, the relative abundance of Fe(III) reducers in them decreased little from levels observed in the pH 6 reactors. Sequences classified within Geobacter, a genus of bacteria known primarily as dissimilatory metal reducers, accounted for 22% and 13.45% of the sequences in the pH 6 and pH 7.5 reactors and only 0.8% of the sequences in the marsh sediment inoculum. In contrast, sequences classified within orders of methanogens were low in abundance, making up only 0.47% and 1.04% of the sequences in the pH 6 and pH 7.5 reactors, respectively. Mass balance calculations demonstrate that the amount of electron donor consumed by each group varied considerably between the sets of reactors. Expressed as a quantity of acetate, the reactions consumed about 160μM of electron donor each in pH 6 reactors. In contrast, methanogenesis consumed over 30 times more electron donor than Fe(III) reduction in the pH 7.5 reactors. Thus, the results of our experiment indicate that the decrease in electron donor consumption by Fe(III) reduction at basic pH was nearly matched by the increase in electron donor consumption by methanogens. Results of geochemical modeling calculations indicate that more energy was available for Fe(III) reduction in the pH 6.0 reactors than the pH 7.5 reactors, matching variation in Fe(III) reduction rates, and that the density of sorbed ferrous iron was higher in pH 6 reactors than pH 7.5 reactors. Thus, the calculation results are consistent with bioenergetics, but not variation in ferrous iron sorption, as a potential mechanism driving variation in the balance between each reaction with pH.

Tidal freshwater wetland soils contain large amounts of organic carbon, some of which is mineralized to carbon dioxide (CO2) and methane (CH4) by a diverse consortium of anaerobic microorganisms that includes fermenters, syntrophs, and methanogens (MG). These microbial groups are tightly linked and often rely on cooperative interspecies metabolisms (i.e., syntrophy) to survive. Environmental perturbations can disrupt these interactions and thus alter the rates and pathways of carbon cycling. One environmental change of particular concern in coastal wetlands is sea level rise, which can result in increased episodic saltwater intrusion events into these ecosystems. These events cause an influx of sulfate (SO4-2) to the soils and may stimulate sulfate-reducing bacteria (SRB), which can directly compete with syntrophs for energy sources (e.g., fermentation products such as butyrate). Since syntroph metabolism generates byproducts that serve as the energy source for many MG, this competition can have indirect negative effects on methanogenesis. In addition, SRB can directly compete with MG for these byproducts, particularly formate, H2, and/or acetate. The goal of this study was to understand how both MG and syntroph-MG consortia respond to and recover from SRB competition during an episodic saltwater intrusion event. To achieve this, microcosms containing soil slurry from a freshwater wetland were subjected to simulated saltwater intrusion, and metabolic inhibitors were used to isolate the activity of the various functional groups. This study focused on the breakdown of butyrate, which is a key energy source in syntroph‑MG consortia metabolisms. The observed changes in butyrate breakdown rates and byproduct accumulation during butyrate degradation assays confirmed that butyrate breakdown was mediated through syntroph-MG consortia, and that formate, rather than H2, was likely used as an electron carrier during syntrophic activity. Additions of SO4‑2 (as Na2SO4) to the freshwater microcosms stimulated SRB activity and shifted the MG community to favor acetoclastic members. These changes were accompanied by a 24% increase in CO2 production and an 80% decrease in CH4 production. Interestingly, when NaCl was added to achieve similar ionic strength, CH4 production decreased by ~32%, suggesting SRB competition is not the only factor affecting methanogenesis. Butyrate degradation rates demonstrated that while SRB were strong competitors for butyrate, concurrent syntrophic metabolism was possible. Further, data show that SRB were poor competitors for acetate, which could explain the increase in acetoclastic MG. Following removal of SRB competition, CH4 production recovered but only by ~50% after 28 days, which suggests that some MG communities in tidal freshwater wetlands may not be resilient to saltwater intrusion events. Over this same time, rates of syntrophic butyrate breakdown largely recovered, but butyrate breakdown resulted in the production of less CH4 and acetate and more CO2 and formate, indicating saltwater intrusion events may lead to persistent changes in the byproducts and pathways of carbon breakdown in tidal freshwater wetlands.

Life on early earth has long been the topic of discussion for many researchers: how did it come to be? Which cells came first? Where can we find them? The most ancient rocks on our planet may hold some of the answers to these questions, but many may only be answered in laboratories. Chemical and morphological traces can be found from Archaean deposits, tantalisingly similar to modern day prokaryotes. Often, they are interpreted as the fossilised remains of bacteria or archaea. However, the caveat remains the abiotic mechanisms with which many similar traces and markers can be formed. The purpose of this thesis was to look into the similarities and differences in abiotic and biological formation of filamentous structures in rocks and observe whether there are chemical or morphological factors that allow for distinguishing between the two. Various laboratory methods were used: chemical gardens to form filamentous abiotic structures and experimental mineralisation of a filamentous methanogen in carbonate, phosphate, and silicate in order to compare and contrast the various mineralisation mechanisms in the fidelity of preservation of the microbes. In the former experiment, analysis with electron paramagnetic resonance (EPR) spectroscopy was carried out to identify potential chemical biomarkers. A combination of scanning and transmission electron microscopy, energy dispersive X-ray (EDX) analysis, X-ray diffraction (XRD) and Raman spectroscopy were also used to analyse the minerals and precipitates formed in both sets of experiments. The results of this research indicate that morphology of filamentous structures and the chemical signatures in biominerals may not be reliable as biogenic indicators. Furthermore, the work on experimental mineralisation reveals the possible biases in the rock record of microbial preservation which is highly dependent on the structure of the cell wall, chemistry of the environment, and the mineral formed. Finally, this work has important outcomes for the search for biomarkers on earth and on other planets and for the recognition of pseudofossils versus microbial fossils in the rock record.