Methanogens are autotrophic Archaea that produce methane as a product of their anaerobic metabolism. They are the largest producers of global methane, contributing over 60% of the total methane budget each year. Methane is an extremely potent greenhouse gas, with emissions providing the second-largest contribution to historical global temperature increases after carbon dioxide. Methanogens have become extremely important industrially as because they are used in the production of biofuels, as well as in treating industrial waste for industrial processes. This report will focus on those successful genetic methods and modifications that have been developed for methanogens and how they have started to contribute to understanding methanogen biochemistry. / text

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Student Number : 9902262G - MSc Dissertation - School of Molecular and Cell Biology - Faculty of Science / Anaerobic fermentative bacteria degrade waste components in landfills where methane (CH4) and carbon dioxide (CO2) are the primary biogases emitted and methanotrophic bacteria in the cover soil oxidise the emitted CH4. Three bi-phasic bench-scale landfill bioreactors were commissioned to evaluate soil nutrient addition effects on CH4 formation and oxidation and to isolate inherent soil methanotrophs using Nitrate Mineral Salts (NMS) medium. Set A soil contained no nutrient additions, Set B soil contained 50 μM nitrate and 150 μM phosphate and Set C soil contained dried sewage cake. Bioreactors were run for a 4 week period and pH, anaerobic gas emissions, volatile fatty acids (VFA), bacterial counts and scanning electron microscopy (SEM) analyses were performed. A pilot study revealed that pH dictated the stability of methanogenesis, where increased VFA levels inhibited methanogenesis. Furthermore, it was revealed that modifications of the NMS medium were needed to enrich for methanotrophs. An in depth study showed that the Set C anaerobic reactor produced the most methane with Set B the least. The hypothesis that methane oxidation in the soil could regulate methane formation in the waste could not be conclusively observed, as a lack of aeration in the soil reactors is believed to have prevented the proliferation of methanotrophs here. No methanotrophs were successfully isolated from soil, but rather major heterotrophic bacterial interference was observed. SEM revealed the presence of rod and cocci forms of bacteria in both leachate and soil, consistent with literature reports, which indicated that the bench-scale landfill bioreactors were capable of promoting bacterial growth.

With consideration to the crude glycerol surplus driven by the growth in biodiesel manufacturing, alternative uses for biodiesel derived glycerol have become increasingly essential. Anaerobic digestion of the glycerol reduces the chemical oxygen demand of the glycerol waste product, while capturing biogas as an energy source that can be used on site to reduce heating cost at a biodiesel facility. In this thesis, kinetic parameters are extracted from batch experimental data and applied to steady state equations. A flow sheet for the application of anaerobic digestion to biodiesel derived glycerol is developed and explored. The economic analysis of the scenario tracks the capital cost, operating cost, and savings associated with implementing the system.

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Our laboratory is interested in studying the junction of glycolysis and the tricarboxylic acid (TCA) cycle, specifically the enzymes phosphoenolpyruvate carboxykinase, pyruvate carboxylase and phosphoenolpyruvate carboxylase. All produce oxaloacetate (OAA) for the cell. OAA production is critical for cell carbon synthesis in the methanogenic archaea. Therefore OAA-generating enzymes are essential for the survival of methanogens. In part of this study we investigated archaeal-type phosphoenolpyruvate carboxylase (PpcA), a new type of phosphoenolpyruvate carboxylase, which is widespread in the archaea and is found in three bacterial species. The form of phosphoenolpyruvate carboxylase (Ppc) that is prevalent in bacteria and plants is not found in the archaea. Due to complications expressing PpcA in the soluble form and difficulty purifying this enzyme from methanogens, an in-depth investigation of this enzyme's biochemical properties has yet to occur. In this study we demonstrate the successful expression of a PpcA homolog in the soluble fraction of Escherichia coli. We purified the recombinant protein to homogeneity. This development provides the means to study the enzyme's biochemical properties and manipulate the primary sequence in order to identify residues critical to the enzyme's function. We also show that this PpcA homolog does have the postulated activity and investigate its biochemical properties. The data show that PpcA has unique properties in regard to the enzyme's substrate and its regulation by metabolites. Our data also reveal that PpcA is a membrane associated protein, unlike Ppc, which is a soluble protein. We also show that pyruvate carboxylase (Pyc) can be expressed recombinantly in Pseudomonas aeruginosa at levels sufficient for structure-function studies. This is a major step forward in the expression in Pyc because it cannot be expressed at high levels in Escherichia coli. These are important developments in studying the enzymes that connect glycolysis and the TCA cycle. / Master of Science

The aim of this thesis was to determine limitation of methanogenesis in restored peatlands and effect of different substrates on potential methane production in restored bog and spruce swamp forest soil. Study sites were located in Šumava National Park in the Czech Republic.

In order to assess the long-term treatment of sulfate- and carbon- rich wastewater at low temperatures, three anaerobic biofilm reactors were operated at 20°C, a hydraulic retention time (HRT)of two days and fed a synthetic wastewater containing lactate and sulfate. The reactors were operated for over 900 days. DNA was extracted from the reactors around days 180 and 800. Three clone libraries, methanogenic archaea (MA), sulfate reducing bacteria (SRB), and bacteria, were constructed and quantitative PCR analysis was performed with the DNA. It was found that anaerobic biofilm reactors can be operated at 20°C with an organic load rate (OLR) of 1.3 g-chemical oxygen demand (COD)/L-day or less and an sulfur load rate (SLR) of 0.2 g-S/L-day with no significant deterioration in process performance. With long acclimation periods, OLR as high as 3.4 g COD/L-d and SLR of 0.3 g/L-d can be tolerated, producing effluent volatile-acid COD levels consistently less than 200 mg/L. Effluent dissolved sulfide and hydrogen sulfide levels were around 600 mg S/L and 150 mg S/L, respectively, during this period. In addition to long term operation, the effect of organic shock loading was assessed. The reactors were able to recover from one but not two lactate spikes of approximately 5,000 mg COD/L. It was determined that long-term stability could be achieved in reactors that contained well balanced, stable populations of lactate- and propionate-degrading SRB and aceticlastic methanogens. Significant populations of fermenters present resulted in an imbalance which caused lactate to be routed through an additional pathway where propionate was formed. Greater numbers of MA than bacteria were found in all reactors. This may be attributed to the availability of acetate in the reactors for MA consumption and to using the immobilized fixed bed reactor type. Aceticlastic methanogens were the dominant methanogen, and were observed to remove nearly all acetate produced in all reactors. SRB were observed to remove lactate in microbially balanced reactors, whereas fermenters degraded lactate in reactors with less balanced populations.

Methanogens are a group of microorganisms that utilize simple compounds such as H₂ + CO₂, acetate and methanol for the production of methane, an end-product of their metabolism.  These obligate anaerobes belonging to the archaeal domain inhabit diverse anoxic environments such as rice paddy fields, human guts, rumen of ruminants, and hydrothermal vents.  In these habitats, methanogens are often exposed to O₂ and previous studies have shown that many methanogens are able to tolerate O2 exposure.  Hence, methanogens must have developed survival strategies to be able to live under oxidative stress conditions.  The anaerobic species that lived on Earth during the early oxygenation event were first to face oxidative stress.  Presumably some of the strategies employed by extant methanogens for combating oxidative stress were developed on early Earth.   Our laboratory is interested in studying the mechanism underlying the oxygen tolerance and oxidative stress responses in methanogenic archaea, which are obligate anaerobe.  Our research concerns two aspects of oxidative stress.  (i) Responses toward extracellular toxic species such as SO32-, that forms as a result of reactions of O₂ with reduced compounds in the environment.  These species are mostly seen in anaerobic environments upon O₂ exposure due to the abundance of reduced components therein.  (ii) Responses toward intracellular toxic species such as superoxide and hydrogen peroxide that are generated upon entry of O₂ and subsequent reaction of O₂ with reduced component inside the cell.  Aerobic microorganisms experience the second problem.  Since a large number of microorganisms of Earth are anaerobes and the oxidative defense mechanisms of anaerobes are relatively less studied, the research in our laboratory has focused on this area.  My thesis research covers two studies that fall in the above-mentioned two focus areas. In 2005-2007 our laboratory discovered that certain methanogens use an unusual sulfite reductase, named F420-dependent sulfite reductase (Fsr), for the detoxification of SO32- that is produced outside the cell from a reaction between oxygen and sulfide.  This reaction occurred during early oxygenation of Earth and continues to occur in deep-sea hydrothermal vents.  Fsr, a flavoprotein, carries out a 6-electron reduction of SO32- to S2-.  It is a chimeric protein where N- and C-terminal halves (Fsr-N and Fsr-C) are homologs of F420H2 dehydrogenase and dissimilatory sulfite reductase (Dsr), respectively.  We hypothesized that Fsr was developed in a methanogen from pre-existing parts.  To begin testing this hypothesis we have carried out bioinformatics analyses of methanogen genomes and found that both Fsr-N homologs and Fsr-C homologs are abundant in methanogens.  We called the Fsr-C homolog dissimilatory sulfite reductase-like protein (Dsr-LP).  Thus, Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP) in methanogens.  During the course of this study, we also identified two new putative F420H2-dependent enzymes, namely F420H2-dependent glutamate synthase and assimilatory sulfite reductase. Another aspect of my research concerns the reactivation of proteins that are deactivated by the entry of oxygen inside the cell.  Here I focused specifically on the role of thioredoxin (Trx) in methanogens.  Trx, a small redox regulatory protein, is ubiquitous in all living cells.  In bacteria and eukarya, Trx regulates a wide variety of cellular processes including cell divison, biosynthesis and oxidative stress response.  Though some Trxs of methanogens have been structurally and biochemically characterized, their physiological roles in these organisms are unknown.  Our bioinformatics analysis suggested that Trx is ubiquitous in methanogens and the pattern of its distribution in various phylogenetic classes paralleled the respective evolutionary histories and metabolic versatilities.  Using a proteomics approach, we have identified 155 Trx targets in a hyperthermophilic phylogenetically deeply-rooted methanogen, Methanocaldococcus jannaschii.  Our analysis of two of these targets employing biochemical assays suggested that Trx is needed for reactivation of oxidatively deactivated enzymes in M. jannaschii.  To our knowledge, this is the first report on the role of Trx in an organism from the archaeal domain. During the course of our work on methanogen Trxs, we investigated the evolutionary histories of different Trx systems that are composed of Trxs and cognate Trx reductases.  In collaboration with other laboratories, we conducted bioinformatics analysis for the distribution of one of such systems, ferredoxin-dependent thioredoxin reductase (FTR), in all organisms.  We found that FTR was most likely originated in the phylogenetically deeply-rooted microaerophilic bacteria where it regulates CO₂ fixation via the reverse citric acid cycle. / Ph. D.