Anaerobic Ciliates and Their Methanogenic Endosymbionts

Anaerobic heterotrichous ciliates (Armophoridae and Clevelandellidae) possess hydrogenosomes that generate molecular hydrogen and ATP. This intracellular source of hydrogen provides the basis for a stable endosymbiotic association with methanogenic archaea. We analyzed the SSU rRNA genes of 18 heterotrichous anaerobic ciliates and their methanogenic endosymbionts in order to unravel the evolution of this mutualistic association. Here, we show that the anaerobic heterotrichous ciliates constitute at least three evolutionary lines. One group consists predominantly of gut-dwelling ciliates, and two to three, potentially four, additional clades comprise ciliates that thrive in freshwater sediments. Their methanogenic endosymbionts belong to only two different taxa that are closely related to free-living methanogenic archaea from the particular ecological niches. The close phylogenetic relationships between the endosymbionts and free-living methanogenic archaea argue for multiple acquisitions from environmental sources, notwithstanding the strictly vertical transmission of the endosymbionts. Since phylogenetic analysis of the small-subunit (SSU) rRNA genes of the hydrogenosomes of these ciliates indicates a descent from the mitochondria of aerobic ciliates, it is likely that anaerobic heterotrichous ciliates hosted endosymbiotic methanogens prior to their radiation. Therefore, our data strongly suggest multiple acquisitions and replacements of endosymbiotic methanogenic archaea during their host's adaptation to the various ecological niches.

Methanogenic endosymbionts are the only known intracellular archaeans and are especially common in anaerobic ciliated protists. Studies on the evolution of associations between anaerobic ciliates and their methanogenic endosymbionts offer an excellent opportunity to broaden our knowledge about symbiosis theory and adaptation of eukaryotes to anoxic environments. Here, the diversity of methanogenic endosymbionts was analyzed with the addition of nine anaerobic ciliate populations that were newly studied by various methods. Results showed that diverse anaerobic ciliates host methanogenic endosymbionts that are limited to a few genera in orders Methanomicrobiales, Methanobacteriales, and Methanosarcinales. For the first time, anaerobic ciliates of the classes Muranotrichea and Prostomatea were found to host methanogenic endosymbionts. Distinct origins of endosymbiosis were revealed for classes Armophorea and Plagiopylea. We posit that armophoreans and plagiopyleans might have harbored Methanoregula (order Methanomicrobiales) and Methanocorpusculum (order Methanomicrobiales), respectively, as methanogenic endosymbionts at the beginning of their evolution. Subsequently, independent endosymbiont replacement events occurred in methanogen-ciliate associations, probably due to ecological transitions, species radiation of ciliate hosts, and vertical transmission bottlenecks of endosymbionts. Our results shed light on the evolution of associations between anaerobic ciliates and methanogens, and identifies the necessary preconditions for illustrating mechanisms by which endosymbioses between these partners were established.Supplementary InformationThe online version contains supplementary material available at 10.1007/s42995-025-00295-9.

Trimyema compressum thrives in anoxic freshwater environments in which it preys on bacteria and grows with fermentative metabolisms. Like many anaerobic protozoa, instead of mitochondria, T. compressum possess hydrogenosomes, which are hydrogen-producing, energy-generating organelles characteristic of anaerobic protozoa and fungi. The cytoplasm of T. compressum harbours hydrogenotrophic methanogens that consume the hydrogen produced by hydrogenosome, which confers an energetic advantage to the host ciliate. Symbiotic associations between methanogenic archaea and Trimyema ciliates are thought to be established independently and/or repeatedly in their evolutional history. In addition to methanogenic symbionts, T. compressum houses bacterial symbiont TC1 whose function is unknown in its cytoplasm. Recently, we analysed whole-genome sequence of TC1 symbiont to investigate its physiological function in the tripartite symbiosis and found that fatty acid synthesis fab operon of TC1 symbiont lacked typical transcriptional repressor, which is normally coded on the upstream of the fab operon. The sequence data suggested that TC1 symbiont contributes to host Trimyema by the synthesis of fatty acid or its derivative. In this review, we summarize the early works and recent progress of the studies on Trimyema ciliates, including a stably cultivable model protozoa T. compressum, and discuss about symbiotic associations in oxygen-scarce environments.

Methanogenic archaea convert bacterial fermentation intermediates from the decomposition of organic material into methane. This process has relevance in the global carbon cycle and finds application in anthropogenic processes, such as wastewater treatment and anaerobic digestion. Furthermore, methanogenic archaea that utilize hydrogen and carbon dioxide as substrates are being employed as biocatalysts for the biomethanation step of power-to-gas technology. This technology converts hydrogen from water electrolysis and carbon dioxide into renewable natural gas (i.e., methane). The application of methanogenic archaea in bioproduction beyond methane has been demonstrated in only a few instances and is limited to mesophilic species for which genetic engineering tools are available. In this chapter, we discuss recent developments for those existing genetically tractable systems and the inclusion of novel genetic tools for thermophilic methanogenic species. We then give an overview of recombinant bioproduction with mesophilic methanogenic archaea and thermophilic non-methanogenic microbes. This is the basis for discussing putative products with thermophilic methanogenic archaea, specifically the species Methanothermobacter thermautotrophicus. We give estimates of potential conversion efficiencies for those putative products based on a genome-scale metabolic model for M. thermautotrophicus.

In situ field experiment shows the potential of methanogenic archaea for biomethane production from underground gas storage in natural rock environment

Effect of operating pressure on direct biomethane production from carbon dioxide and exogenous hydrogen in the anaerobic digestion of sewage sludge

Methane hydrate exists in huge amounts in certain locations, in sea sediments and the geological structures below them, at low temperature and high pressure. Production methods are in development to produce the methane to a floating platform. There it can be reformed to produce hydrogen and carbon dioxide, in an endothermic process. Some of the methane can be burned to provide heat energy to develop all needed power on the platform and to support the reforming process. After separation, the hydrogen is the valuable and transportable product. All carbon dioxide produced on the platform can be separated from other gases and then sequestered in the sea as carbon dioxide hydrate. In this way, hydrogen is made available without the release of carbon dioxide to the atmosphere, and the hydrogen could be an enabling step toward a world hydrogen economy.

Many anaerobic ciliated protozoa contain organelles of mitochondrial ancestry called hydrogenosomes. These organelles generate molecular hydrogen that is consumed by methanogenic Archaea, living in endosymbiosis within many of these ciliates. Here we describe a new species of anaerobic ciliate, Trimyema finlayi n. sp., by using silver impregnation and microscopy to conduct a detailed morphometric analysis. Comparisons with previously published morphological data for this species, as well as the closely related species, Trimyema compressum, demonstrated that despite them being similar, both the mean cell size and the mean number of somatic kineties are lower for T. finlayi than for T. compressum, which suggests that they are distinct species. This was also supported by analysis of the 18S rRNA genes from these ciliates, the sequences of which are 97.5% identical (6 substitutions, 1479 compared bases), and in phylogenetic analyses these sequences grouped with other 18S rRNA genes sequenced from previous isolates of the same respective species. Together these data provide strong evidence that T. finlayi is a novel species of Trimyema, within the class Plagiopylea. Various microscopic techniques demonstrated that T. finlayi n. sp. contains polymorphic endosymbiotic methanogens, and analysis of the endosymbionts’ 16S rRNA gene showed that they belong to the genus Methanocorpusculum, which was confirmed using fluorescence in situ hybridization with specific probes. Despite the degree of similarity and close relationship between these ciliates, T. compressum contains endosymbiotic methanogens from a different genus, Methanobrevibacter. In phylogenetic analyses of 16S rRNA genes, the Methanocorpusculum endosymbiont of T. finlayi n. sp. grouped with sequences from Methanomicrobia, including the endosymbiont of an earlier isolate of the same species, ‘Trimyema sp.,’ which was sampled approximately 22 years earlier, at a distant (∼400 km) geographical location. Identification of the same endosymbiont species in the two separate isolates of T. finlayi n. sp. provides evidence for spatial and temporal stability of the Methanocorpusculum–T. finlayi n. sp. endosymbiosis. T. finlayi n. sp. and T. compressum provide an example of two closely related anaerobic ciliates that have endosymbionts from different methanogen genera, suggesting that the endosymbionts have not co-speciated with their hosts.

Contains fulltext : 223983.pdf (Publisher’s version ) (Open Access)

Reduction in carbon dioxide and production of methane by biological reaction in the electronics industry

The association between anaerobic ciliates and methanogenic archaea has been recognized for over a century. Nevertheless, knowledge of these associations is limited to a few ciliate species, and so the identification of patterns of host-symbiont specificity has been largely speculative. In this study, we integrated microscopy and genetic identification to survey the methanogenic symbionts of 32 free-living anaerobic ciliate species, mainly from the order Metopida. Based on Sanger and Illumina sequencing of the 16S rRNA gene, our results show that a single methanogenic symbiont population, belonging to Methanobacterium, Methanoregula, or Methanocorpusculum, is dominant in each host strain. Moreover, the host's taxonomy (genus and above) and environment (i.e. endobiotic, marine/brackish, or freshwater) are linked with the methanogen identity at the genus level, demonstrating a strong specificity and fidelity in the association. We also established cultures containing artificially co-occurring anaerobic ciliate species harboring different methanogenic symbionts. This revealed that the host-methanogen relationship is stable over short timescales in cultures without evidence of methanogenic symbiont exchanges, although our intraspecific survey indicated that metopids also tend to replace their methanogens over longer evolutionary timescales. Therefore, anaerobic ciliates have adapted a mixed transmission mode to maintain and replace their methanogenic symbionts, allowing them to thrive in oxygen-depleted environments.

Trimyema ciliates thrive in various anoxic environments in which they prey on bacteria and grow with fermentative metabolisms. Like many anaerobic protozoa, instead of mitochondria, Trimyema possess hydrogenosomes, which are hydrogen-producing, energy-generating organelles characteristic of anaerobic protozoa and fungi. The cytoplasm of Trimyema harbours hydrogenotrophic methanogens that consume the hydrogen produced by these organelles, which confers an energetic advantage to the host ciliate. Symbiotic associations between methanogenic archaea and Trimyema ciliates are thought to be established independently and/or repeatedly in their evolutional history. In addition to methanogenic symbionts, it has been shown that Trimyema compressum houses bacterial symbionts. Although almost nothing is known about the symbionts except for their phylogeny, this intriguing multi-symbiosis would be a good model for investigating symbiotic interactions among bacteria, archaea, and eukaryotes. In this chapter, we summarise the early works and recent progress of studies on Trimyema ciliates, in particular T. compressum, and discuss the nature of this symbiosis.

Methane is the second most important greenhouse gas on earth. It is produced by methanogenic archaea, which play an important role in the global carbon cycle. Three main methanogenesis pathways are known: in the hydrogenotrophic pathway H2 and carbon dioxide are used for methane production, whereas in the methylotrophic pathway small methylated carbon compounds like methanol and methylated amines are used. In the aceticlastic pathway, acetate is disproportionated to methane and carbon dioxide. However, next to these conventional substrates, further methanogenic substrates and pathways have been discovered. Several phylogenetically distinct methanogenic lineages (Methanosphaera, Methanimicrococcus, Methanomassiliicoccus, Methanonatronarchaeum) have evolved hydrogen-dependent methylotrophic methanogenesis without the ability to perform either hydrogenotrophic or methylotrophic methanogenesis. Genome analysis of the deep branching Methanonatronarchaeum revealed an interesting membrane-bound hydrogenase complex affiliated with the hardly described class 4 g of multisubunit hydrogenases possibly providing reducing equivalents for anabolism. Furthermore, methylated sulfur compounds such as methanethiol, dimethyl sulfide, and methylmercaptopropionate were described to be converted into adapted methylotrophic methanogenesis pathways of Methanosarcinales strains. Moreover, recently it has been shown that the methanogen Methermicoccus shengliensis can use methoxylated aromatic compounds in methanogenesis. Also, tertiary amines like choline (N,N,N-trimethylethanolamine) or betaine (N,N,N-trimethylglycine) have been described as substrates for methane production in Methanococcoides and Methanolobus strains. This review article will provide in-depth information on genome-guided metabolic reconstructions, physiology, and biochemistry of these unusual methanogenesis pathways.Key points• Newly discovered methanogenic substrates and pathways are reviewed for the first time.• The review provides an in-depth analysis of unusual methanogenesis pathways.• The hydrogenase complex of the deep branching Methanonatronarchaeum is analyzed.

Methanogenesis is the biological production of methane and is utilized by methanogenic archaea (methanogens) as a form of anaerobic respiration to generate energy. Methanogens are found in virtually any anaerobic environment and are responsible for over 70% of total atmospheric methane, a greenhouse gas 84 times more potent than carbon dioxide over a twenty-year period. Due to the abundance of methane and its powerful heat-trapping capability, it is among the most significant greenhouse gases. In addition to methane's importance as a greenhouse gas, it is a valuable and clean energy source that emits 50% less carbon dioxide than coal combustion. An attractive possibility for further exploiting the chemical energy stored in methane is to convert it to more useable liquid fuels and other value-added chemicals. This could be accomplished by producing a methanogenic strain capable of reversing methanogenesis and converting methane to methanol. Before this can be realized, however, the chemistry of methanogenesis must be more fully understood. The final, rate-determining, methane-forming step of methanogenesis is catalyzed by methyl-coenzyme M reductase (MCR) and its prosthetic group, cofactor F430, a unique nickel-containing tetrapyrrole that is the essential catalytic component of MCR. Recently, multiple F430 variants have been discovered in several methanogenic species, including the two model methanogens Methanococcus maripaludis and Methanosarcina acetivorans. Our research suggests that one of these variants, mercaptopropionate-F430, (F430-3) is produced by M. maripaludis almost exclusively in stationary phase of growth, suggesting that nutrient deprivation induces the production of this variant. We hypothesize that hydrogen deprivation is involved in inducing this expression, but future experiments will be conducted to confirm this. We further have implicated a likely enzyme involved in the first step of F430-3 biosynthesis, the insertion of a sulfur atom. This research sets the stage for further investigation into how these two variants modulate MCR catalysis.

Alcohol Production from Carbon Dioxide: Methanol as a Fuel and Chemical Feedstock