Physiological and genomic characterization of thermophilic methanotrophic archaea and their partner-bacteria

Methane is a potent greenhouse gas and its atmospheric concentration is strongly influenced by microbial processes. In anoxic marine environments 80% of the methane is oxidized by anaerobic microorganisms leading to reduced oceanic methane emissions. This anaerobic oxidation of methane (AOM) is coupled to sulfate reduction and is mediated by microbial consortia of anaerobic methane-oxidizing archaea and partner bacteria. The physiology of the consortia is incompletely understood but is thought to base on a metabolic interdependency of the partners, a syntrophy. The research presented in this PhD thesis focused on the physiology and genomic profile of AOM consortia, in particular on the microorganisms that are active at elevated temperatures (thermophiles). The thermophilic AOM is performed by a unique consortium of ANME-1 archaea and HotSeep-1 bacteria. In Chapter II we describe physiological studies and gene expression experiments with thermophilic AOM consortia and propose a syntrophy of AOM via direct exchange of reducing equivalents. In support of this hypothesis we visualized cell-to-cell connections in these consortia that we suggest to function as conductive nanowires in interspecies electron transfer. For the thermophilic bacterial partner, HotSeep-1 we obtained an ANME-1-free enrichment culture using hydrogen as alternative energy source, and by physiological and genomic investigation we show in Chapter III that this bacterial partner grows as chemolithoautotrophic sulfate reducer. Based on phylogenetic analysis we propose that HotSeep-1 presents a novel species, Candidatus Desulfofervidus auxilii. ANME-1, the archaeon participating in thermophilic AOM, belongs to a large group of uncultured organisms, which are known to have reversed the methanogenesis pathway to metabolize methane. The metabolic diversity among members of the ANME-1 group is still widely unexplored. In a comparative genome analysis of different ANME-1 in Chapter IV we show central aspects of their metabolism including a modified reverse methanogenesis pathway and abundant cytochromes possibly relevant for electron transfer. Environments of AOM activity and in vitro AOM enrichments are dominated by AOM consortia, but other microorganisms sustain as low abundant community whose function is not well understood. In Chapter V we show the cultivation of methanogens and sulfur-disproportionating bacteria from AOM enrichments. In conclusion the work of this PhD thesis has advanced our understanding of the functioning of thermophilic AOM, while further detailed comparative approaches are necessary to comprehend AOM syntrophy in all its detail and diversity

Physiological and genomic characterization of thermophilic methanotrophic archaea and partner bacteria

Methane is a potent greenhouse gas and its atmospheric concentration is strongly influenced by microbial processes. In anoxic marine environments 80% of the methane is oxidized by anaerobic microorganisms leading to reduced oceanic methane emissions. This anaerobic oxidation of methane (AOM) is coupled to sulfate reduction and is mediated by microbial consortia of anaerobic methane-oxidizing archaea and partner bacteria. The physiology of the consortia is incompletely understood but is thought to base on a metabolic interdependency of the partners, a syntrophy. The research presented in this PhD thesis focused on the physiology and genomic profile of AOM consortia, in particular on the microorganisms that are active at elevated temperatures (thermophiles). The thermophilic AOM is performed by a unique consortium of ANME-1 archaea and HotSeep-1 bacteria. In Chapter II we describe physiological studies and gene expression experiments with thermophilic AOM consortia and propose a syntrophy of AOM via direct exchange of reducing equivalents. In support of this hypothesis we visualized cell-to-cell connections in these consortia that we suggest to function as conductive nanowires in interspecies electron transfer. For the thermophilic bacterial partner, HotSeep-1 we obtained an ANME-1-free enrichment culture using hydrogen as alternative energy source, and by physiological and genomic investigation we show in Chapter III that this bacterial partner grows as chemolithoautotrophic sulfate reducer. Based on phylogenetic analysis we propose that HotSeep-1 presents a novel species, Candidatus Desulfofervidus auxilii. ANME-1, the archaeon participating in thermophilic AOM, belongs to a large group of uncultured organisms, which are known to have reversed the methanogenesis pathway to metabolize methane. The metabolic diversity among members of the ANME-1 group is still widely unexplored. In a comparative genome analysis of different ANME-1 in Chapter IV we show central aspects of their metabolism including a modified methanogenesis pathway and abundant cytochromes possibly relevant for electron transfer. Environments of AOM activity and in vitro AOM enrichments are dominated by AOM consortia, but other microorganisms sustain as low abundant community whose function is not well understood. In Chapter V we show the cultivation of methanogens and sulfur disproportionating bacteria from AOM enrichments. In conclusion the work of this PhD thesis has advanced our understanding of the functioning of thermophilic AOM, while further detailed comparative approaches are necessary to comprehend AOM syntrophy in all its detail and diversity

Establishing anaerobic hydrocarbon-degrading enrichment cultures of microorganisms under strictly anoxic conditions

Traditionally, the description of microorganisms starts with their isolation from an environmental sample. Many environmentally relevant anaerobic microorganisms grow very slowly, and often they rely on syntrophic interactions with other microorganisms. This impedes their isolation and characterization by classic microbiological techniques. We developed and applied an approach for the successive enrichment of syntrophic hydrocarbon-degrading microorganisms from environmental samples. We collected samples from microbial mat-covered hydrothermally heated hydrocarbon-rich sediments of the Guaymas Basin and mixed them with synthetic mineral medium to obtain sediment slurries. Supplementation with defined substrates (i.e., methane or butane), incubation at specific temperatures, and a regular maintenance procedure that included the measurement of metabolic products and stepwise dilutions enabled us to establish highly active, virtually sediment-free enrichment cultures of actively hydrocarbon-degrading communities in a 6-months to several-years' effort. Using methane as sole electron donor shifted the originally highly diverse microbial communities toward defined mixed cultures dominated by syntrophic consortia consisting of anaerobic methane-oxidizing archaea (ANME) and different sulfate-reducing bacteria. Cultivation with butane at 50 °C yielded consortia of archaea belonging to Candidatus Syntrophoarchaeum and Candidatus Desulfofervidus auxilii partner bacteria. This protocol also describes sampling for further molecular characterization of enrichment cultures by fluorescence in situ hybridization (FISH), and transcriptomics and metabolite analyses, which can provide insights into the functioning of hydrocarbon metabolism in archaea and resolve important mechanisms that enable electron transfer to their sulfate-reducing partner bacteria

Physiologische und genomische Beschreibung von thermophilen methanotrophen Archaeen und ihren Partner-Bakterien

Methane is a potent greenhouse gas and its atmospheric concentration is strongly influenced by microbial processes. In anoxic marine environments 80% of the methane is oxidized by anaerobic microorganisms leading to reduced oceanic methane emissions. This anaerobic oxidation of methane (AOM) is coupled to sulfate reduction and is mediated by microbial consortia of anaerobic methane-oxidizing archaea and partner bacteria. The physiology of the consortia is incompletely understood but is thought to base on a metabolic interdependency of the partners, a syntrophy. The research presented in this PhD thesis focused on the physiology and genomic profile of AOM consortia, in particular on the microorganisms that are active at elevated temperatures (thermophiles). The thermophilic AOM is performed by a unique consortium of ANME-1 archaea and HotSeep-1 bacteria. In Chapter II we describe physiological studies and gene expression experiments with thermophilic AOM consortia and propose a syntrophy of AOM via direct exchange of reducing equivalents. In support of this hypothesis we visualized cell-to-cell connections in these consortia that we suggest to function as conductive nanowires in interspecies electron transfer. For the thermophilic bacterial partner, HotSeep-1 we obtained an ANME-1-free enrichment culture using hydrogen as alternative energy source, and by physiological and genomic investigation we show in Chapter III that this bacterial partner grows as chemolithoautotrophic sulfate reducer. Based on phylogenetic analysis we propose that HotSeep-1 presents a novel species, Candidatus Desulfofervidus auxilii. ANME-1, the archaeon participating in thermophilic AOM, belongs to a large group of uncultured organisms, which are known to have reversed the methanogenesis pathway to metabolize methane. The metabolic diversity among members of the ANME-1 group is still widely unexplored. In a comparative genome analysis of different ANME-1 in Chapter IV we show central aspects of their metabolism including a modified reverse methanogenesis pathway and abundant cytochromes possibly relevant for electron transfer. Environments of AOM activity and in vitro AOM enrichments are dominated by AOM consortia, but other microorganisms sustain as low abundant community whose function is not well understood. In Chapter V we show the cultivation of methanogens and sulfur-disproportionating bacteria from AOM enrichments. In conclusion the work of this PhD thesis has advanced our understanding of the functioning of thermophilic AOM, while further detailed comparative approaches are necessary to comprehend AOM syntrophy in all its detail and diversity

Culexarchaeia, a novel archaeal class of anaerobic generalists inhabiting geothermal environments

Abstract Geothermal environments, including terrestrial hot springs and deep-sea hydrothermal sediments, often contain many poorly understood lineages of archaea. Here, we recovered ten metagenome-assembled genomes (MAGs) from geothermal sediments and propose that they constitute a new archaeal class within the TACK superphylum, “Candidatus Culexarchaeia”, named after the Culex Basin in Yellowstone National Park. Culexarchaeia harbor distinct sets of proteins involved in key cellular processes that are either phylogenetically divergent or are absent from other closely related TACK lineages, with a particular divergence in cell division and cytoskeletal proteins. Metabolic reconstruction revealed that Culexarchaeia have the capacity to metabolize a wide variety of organic and inorganic substrates. Notably, Culexarchaeia encode a unique modular, membrane associated, and energy conserving [NiFe]-hydrogenase complex that potentially interacts with heterodisulfide reductase (Hdr) subunits. Comparison of this [NiFe]-hydrogenase complex with similar complexes from other archaea suggests that interactions between membrane associated [NiFe]-hydrogenases and Hdr may be more widespread than previously appreciated in both methanogenic and non-methanogenic lifestyles. The analysis of Culexarchaeia further expands our understanding of the phylogenetic and functional diversity of lineages within the TACK superphylum and the ecology, physiology, and evolution of these organisms in extreme environments

Diversity and function of methyl-coenzyme M reductase-encoding archaea in Yellowstone hot springs revealed by metagenomics and mesocosm experiments

Abstract Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of methyl-coenzyme M reductase subunit A (mcrA) gene amplicons demonstrated a wide diversity of Mcr-encoding archaea inhabit geothermal features with differing physicochemical regimes across YNP. From three selected hot springs we recovered twelve Mcr-encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus (Ca.) Methanomethylicia, Ca. Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represent minor fractions of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in the stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca. Korarchaeia, Ca. Nezhaarchaeia, and Archaeoglobi. We revealed that diverse Mcr-encoding archaea with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP and can be enriched under methanogenic conditions. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond

Anaerobic Methane Oxidizers

The anaerobic oxidation of methane (AOM) with sulfate as the final electron acceptor according to the net reaction CH4 + SO42- -> HCO3- -> HS- + H2O is the major sink of methane in the ocean floor and hence a significant process in the marine methane budget and the global carbon cycle. Since its discovery, much has been learned about the distribution of the AOM process, its activity in different settings, and connections to other metabolic reactions in the seafloor. AOM is performed by consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). Since all known ANME and most of their partner bacteria have so far resisted isolation, the physiology of both organisms has been largely inferred from culture-independent approaches on natural enrichments or enrichment cultures. All known ANME are related to methanogenic Euryarchaeota, and as such they reverse the methanogenesis pathway to activate and completely oxidize methane. The reducing equivalents are shuttled to the partner bacteria, which use them for sulfate reduction. Recently, evidence has been found for ANME that can use nitrate or iron as electron acceptors. The exact mechanisms for the required exchange of reducing equivalents in AOM and their genetic codes are yet poorly understood, but recently discovered accumulations of cytochromes and nanowire connections in the intercellular space of the consortia suggest direct electron transfer between both partners

Thermophilic archaea activate butane via alkyl-CoM formation

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C1-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C4 hydrocarbon butane. The archaea, proposed genus ‘Candidatus Syntrophoarchaeum’, show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding β-oxidation enzymes, carbon monoxide dehydrogenase and reversible C1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons

Anaerobic Methane Oxidizers

The anaerobic oxidation of methane (AOM) with sulfate as the final electron acceptor according to (CH4 + SO4 2− → HCO3 − + HS− + H2O) is the major sink of methane in the oceans and hence a significant process in the global carbon cycle and methane budget. Anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) are assumed to act as a syntrophic consortium where the archaeal partner activates and metabolizes methane, leading to an intermediate that is scavenged as electron donor by the sulfate-reducing partner. All known anaerobic methanotrophs are related to the methanogenic Euryarchaeota. Recently, much has been learned about the distribution, activity, and physiology of the ANME, however, not a single member of these groups has been obtained in culture and the biochemical functioning of AOM remains unknown