Methane production from protozoan endosymbionts following stimulation of microbial metabolism within subsurface sediments
TL;DR: Results suggest that, following the stimulation of subsurface microbial growth with acetate, protozoa harboring methanogenic endosymbionts become important members of the microbial community, feeding on moribund biomass and producing methane.
Abstract: Previous studies have suggested that protozoa prey on Fe(III)- and sulfate-reducing bacteria that are enriched when acetate is added to uranium contaminated subsurface sediments to stimulate U(VI) reduction. In order to determine whether protozoa continue to impact subsurface biogeochemistry after these acetate amendments have stopped, 18S rRNA and s-tubulin sequences from this phase of an in situ uranium bioremediation field experiment were analyzed. Sequences most similar to Metopus species predominated, with the majority of sequences most closely related to M. palaeformis, a cilitated protozoan known to harbor methanogenic symbionts. Quantification of mcrA mRNA transcripts in the groundwater suggested that methanogens closely related to Metopus endosymbionts were metabolically active at this time. There was a strong correlation between the number of mcrA transcripts from the putative endosymbiotic methanogen and Metopus s-tubulin mRNA transcripts during the course of the field experiment, suggesting that the activity of the methanogens was dependent upon the activity of the Metopus species. Addition of the eukaryotic inhibitors cyclohexamide and colchicine to laboratory incubations of acetate-amended subsurface sediments significantly inhibited methane production and there was a direct correlation between methane concentration and Metopus s-tubulin and putative symbiont mcrA gene copies. These results suggest that, following the stimulation of subsurface microbial growth with acetate, protozoa harboring methanogenic endosymbionts become important members of the microbial community, feeding on moribund biomass and producing methane.
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TL;DR: This study sequenced DNA from complex sediment and planktonic consortia from an aquifer adjacent to the Colorado River and reconstructed the first complete genomes for Archaea using cultivation-independent methods, which dramatically expand genomic sampling of the domain Archaea and clarify taxonomic designations within a major superphylum.
463 citations
Cites background from "Methane production from protozoan e..."
...Although evidence for their activity has been detected during in situ acetate amendment [27], methanogenic Archaea did not appear to be abundant in the filtrate....
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TL;DR: The results suggest that the reason that Geobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with Methanothrix spp.
Abstract: The possibility that Methanothrix (formerly Methanosaeta) and Geobacter species cooperate via direct interspecies electron transfer (DIET) in terrestrial methanogenic environments was investigated in rice paddy soils. Genes with high sequence similarity to the gene for the PilA pilin monomer of the electrically conductive pili (e-pili) of Geobacter sulfurreducens accounted for over half of the PilA gene sequences in metagenomic libraries and 42% of the mRNA transcripts in RNA sequencing (RNA-seq) libraries. This abundance of e-pilin genes and transcripts is significant because e-pili can serve as conduits for DIET. Most of the e-pilin genes and transcripts were affiliated with Geobacter species, but sequences most closely related to putative e-pilin genes from genera such as Desulfobacterium, Deferribacter, Geoalkalibacter, and Desulfobacula, were also detected. Approximately 17% of all metagenomic and metatranscriptomic bacterial sequences clustered with Geobacter species, and the finding that Geobacter spp. were actively transcribing growth-related genes indicated that they were metabolically active in the soils. Genes coding for e-pilin were among the most highly transcribed Geobacter genes. In addition, homologs of genes encoding OmcS, a c-type cytochrome associated with the e-pili of G. sulfurreducens and required for DIET, were also highly expressed in the soils. Methanothrix species in the soils highly expressed genes for enzymes involved in the reduction of carbon dioxide to methane. DIET is the only electron donor known to support CO2 reduction in Methanothrix Thus, these results are consistent with a model in which Geobacter species were providing electrons to Methanothrix species for methane production through electrical connections of e-pili.IMPORTANCEMethanothrix species are some of the most important microbial contributors to global methane production, but surprisingly little is known about their physiology and ecology. The possibility that DIET is a source of electrons for Methanothrix in methanogenic rice paddy soils is important because it demonstrates that the contribution that Methanothrix makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that when Methanothrix species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus, Methanothrix growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason that Geobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with Methanothrix spp., and possibly other methanogens, via DIET.
229 citations
Cites methods from "Methane production from protozoan e..."
...Sulfate reduction was monitored with an ion chromatograph (ICS-2100; Dionex, CA) equipped with an AS18 column under isocratic elution with 32 mM KOH as the eluent (57)....
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TL;DR: The current understanding of the archaeome in humans, the specific adaptations involved in interaction with the resident microbial community as well as with the host, and the roles of the archaeological community in both health and disease are presented.
Abstract: Host-associated microbial communities have an important role in shaping the health and fitness of plants and animals. Most studies have focused on the bacterial, fungal or viral communities, but often the archaeal component has been neglected. The archaeal community, the so-called archaeome, is now increasingly recognized as an important component of host-associated microbiomes. It is composed of various lineages, including mainly Methanobacteriales and Methanomassiliicoccales (Euryarchaeota), as well as representatives of the Thaumarchaeota. Host–archaeome interactions have mostly been delineated from methanogenic archaea in the gastrointestinal tract, where they contribute to substantial methane production and are potentially also involved in disease-relevant processes. In this Review, we discuss the diversity and potential roles of the archaea associated with protists, plants and animals. We also present the current understanding of the archaeome in humans, the specific adaptations involved in interaction with the resident microbial community as well as with the host, and the roles of the archaeome in both health and disease. The archaeal community, the archaeome, is now increasingly recognized as an important component of host-associated microbiomes. In this Review, Moissl-Eichinger and colleagues discuss the diversity and potential roles of the archaea associated with protists, plants and animals, highlighting the potential roles of archaea in human health and disease.
106 citations
12 Feb 2019
TL;DR: Fermentation in a semi-continuous in-vitro rumen system suggests that A. taxiformis can reduce methane production from enteric fermentation in dairy cattle by 95% when added at a 5% OM inclusion rate without any obvious negative impacts on volatile fatty acid production.
Abstract: Recent studies using batch-fermentation suggest that the red macroalgae Asparagopsis taxiformis has the potential to reduce methane (CH4) production from beef cattle by up to ~ 99% when added to Rhodes grass hay; a common feed in the Australian beef industry. These experiments have shown significant reductions in CH4 without compromising other fermentation parameters (i.e. volatile fatty acid production) with A. taxiformis organic matter (OM) inclusion rates of up to 5%. In the study presented here, A. taxiformis was evaluated for its ability to reduce methane production from dairy cattle fed a mixed ration widely utilized in California, the largest milk producing state in the US. Fermentation in a semi-continuous in-vitro rumen system suggests that A. taxiformis can reduce methane production from enteric fermentation in dairy cattle by 95% when added at a 5% OM inclusion rate without any obvious negative impacts on volatile fatty acid production. High-throughput 16S ribosomal RNA (rRNA) gene amplicon sequencing showed that seaweed amendment effects rumen microbiome consistent with the Anna Karenina hypothesis, with increased β-diversity, over time scales of approximately 3 days. The relative abundance of methanogens in the fermentation vessels amended with A. taxiformis decreased significantly compared to control vessels, but this reduction in methanogen abundance was only significant when averaged over the course of the experiment. Alternatively, significant reductions of CH4 in the A. taxiformis amended vessels was measured in the early stages of the experiment. This suggests that A. taxiformis has an immediate effect on the metabolic functionality of rumen methanogens whereas its impact on microbiome assemblage, specifically methanogen abundance, is delayed. The methane reducing effect of A. taxiformis during rumen fermentation makes this macroalgae a promising candidate as a biotic methane mitigation strategy for dairy cattle. But its effect in-vivo (i.e. in dairy cattle) remains to be investigated in animal trials. Furthermore, to obtain a holistic understanding of the biochemistry responsible for the significant reduction of methane, gene expression profiles of the rumen microbiome and the host animal are warranted.
86 citations
Cites background from "Methane production from protozoan e..."
...It is well known that there is a mutualistic relationship between protozoa and methanogens [33, 34], and it has been shown before that the removal of rumen protozoa results in a reduction of the methanogen population and methanogenesis during enteric fermentation [35, 36]....
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TL;DR: These studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea, and suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the ext racellular environment.
Abstract: Extracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew Methanosarcina acetivorans with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, c-type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in Archaea. IMPORTANCE The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea was previously proposed, but these studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea. The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens but possibly also for Archaea that anaerobically oxidize methane.
63 citations
Cites methods from "Methane production from protozoan e..."
...Methane in the headspace was measured by gas chromatography with a flame ionization detector (Shimadzu, GC-8A) as previously described (85)....
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References
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TL;DR: In this paper, a new mathematical method for estimating the number of transitional and transversional substitutions per site, as well as the total number of nucleotide substitutions was proposed, taking into account excess transitions, unequal nucleotide frequencies, and variation of substitution rate among different sites.
Abstract: Examining the pattern of nucleotide substitution for the control region of mitochondrial DNA ( mtDNA ) in humans and chimpanzees, we developed a new mathematical method for estimating the number of transitional and transversional substitutions per site, as well as the total number of nucleotide substitutions. In this method, excess transitions, unequal nucleotide frequencies, and variation of substitution rate among different sites are all taken into account. Application of this method to human and chimpanzee data suggested that the transition / transversion ratio for the entire control region was - 15 and nearly the same for the two species. The 95% confidence interval of the age of the common ancestral mtDNA was estimated to be 80,000-480,000 years in humans and 0.57-2.72 Myr in common chimpanzees.
9,144 citations
TL;DR: A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolation of the 16 S rRNA or cloning of its gene, and its phylogenetic usefulness is evaluated by examination of several 17S rRNAs whose gene sequences are known.
Abstract: Although the applicability of small subunit ribosomal RNA (16S rRNA) sequences for bacterial classification is now well accepted, the general use of these molecules has been hindered by the technical difficulty of obtaining their sequences. A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolation of the 16S rRNA or cloning of its gene. The 16S rRNA in bulk cellular RNA preparations is selectively targeted for dideoxynucleotide-terminated sequencing by using reverse transcriptase and synthetic oligodeoxynucleotide primers complementary to universally conserved 16S rRNA sequences. Three particularly useful priming sites, which provide access to the three major 16S rRNA structural domains, routinely yield 800-1000 nucleotides of 16S rRNA sequence. The method is evaluated with respect to accuracy, sensitivity to modified nucleotides in the template RNA, and phylogenetic usefulness, by examination of several 16S rRNAs whose gene sequences are known. The relative simplicity of this approach should facilitate a rapid expansion of the 16S rRNA sequence collection available for phylogenetic analyses.
3,006 citations
"Methane production from protozoan e..." refers methods in this paper
...Gene fragments from the bacterial 16S rRNA gene were amplified with 8F (Eden et al., 1991) and 519R (Lane et al., 1985); 344F and 915R (Casamayor et al., 2002) amplified archaeal 16S rRNA gene fragments; 515F (Giovannoni et al., 1988) and 1209R (Reysenbach et al., 1992) amplified eukaryotic 18S…...
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TL;DR: This is the first demonstration that microorganisms can completely oxidize organic compounds with Fe(III) or Mn(IV) as the sole electron acceptor and that oxidation of organic matter coupled to dissimilatory Fe( III), Mn( IV), or Mn (IV) reduction can yield energy for microbial growth.
Abstract: A dissimilatory Fe(III)- and Mn(IV)-reducing microorganism was isolated from freshwater sediments of the Potomac River, Maryland. The isolate, designated GS-15, grew in defined anaerobic medium with acetate as the sole electron donor and Fe(III), Mn(IV), or nitrate as the sole electron acceptor. GS-15 oxidized acetate to carbon dioxide with the concomitant reduction of amorphic Fe(III) oxide to magnetite (Fe(3)O(4)). When Fe(III) citrate replaced amorphic Fe(III) oxide as the electron acceptor, GS-15 grew faster and reduced all of the added Fe(III) to Fe(II). GS-15 reduced a natural amorphic Fe(III) oxide but did not significantly reduce highly crystalline Fe(III) forms. Fe(III) was reduced optimally at pH 6.7 to 7 and at 30 to 35 degrees C. Ethanol, butyrate, and propionate could also serve as electron donors for Fe(III) reduction. A variety of other organic compounds and hydrogen could not. MnO(2) was completely reduced to Mn(II), which precipitated as rhodochrosite (MnCO(3)). Nitrate was reduced to ammonia. Oxygen could not serve as an electron acceptor, and it inhibited growth with the other electron acceptors. This is the first demonstration that microorganisms can completely oxidize organic compounds with Fe(III) or Mn(IV) as the sole electron acceptor and that oxidation of organic matter coupled to dissimilatory Fe(III) or Mn(IV) reduction can yield energy for microbial growth. GS-15 provides a model for how enzymatically catalyzed reactions can be quantitatively significant mechanisms for the reduction of iron and manganese in anaerobic environments.
2,233 citations
"Methane production from protozoan e..." refers methods in this paper
...…spectrophotometer (Spectronic Genosys2; Thermo Electron Corp., Mountain View, CA) at an absorbance of 562 nm Frontiers in Microbiology | Terrestrial Microbiology August 2014 | Volume 5 | Article 366 | 2 after a 1 h extraction with 0.5 N HCl (Lovley et al., 1987; Lovley and Phillips, 1988)....
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TL;DR: It is shown that anaerobic metal-reducing bacteria can play a key role in the mobilization of arsenic in sediments collected from a contaminated aquifer in West Bengal and that, for the sediments in this study, arsenic release took place after Fe(iii) reduction, rather than occurring simultaneously.
Abstract: The contamination of ground waters, abstracted for drinking and irrigation, by sediment-derived arsenic threatens the health of tens of millions of people worldwide, most notably in Bangladesh and West Bengal1,2,3. Despite the calamitous effects on human health arising from the extensive use of arsenic-enriched ground waters in these regions, the mechanisms of arsenic release from sediments remain poorly characterized and are topics of intense international debate4,5,6,7,8. We use a microscosm-based approach to investigate these mechanisms: techniques of microbiology and molecular ecology are used in combination with aqueous and solid phase speciation analysis of arsenic. Here we show that anaerobic metal-reducing bacteria can play a key role in the mobilization of arsenic in sediments collected from a contaminated aquifer in West Bengal. We also show that, for the sediments in this study, arsenic release took place after Fe(iii) reduction, rather than occurring simultaneously. Identification of the critical factors controlling the biogeochemical cycling of arsenic is one important contribution to fully informing the development of effective strategies to manage these and other similar arsenic-rich ground waters worldwide.
1,153 citations
"Methane production from protozoan e..." refers background in this paper
...…groundwater, arsenic was also released (Giloteaux et al., 2013), presumably as the result of microbial reduction of Fe(III) minerals that adsorb arsenic in the subsurface (Dowdle et al., 1996; Redman et al., 2002; Islam et al., 2004; Rowland et al., 2007; Hery et al., 2010; Giloteaux et al., 2013)....
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TL;DR: The transfer of hydrogen and formate between bacteria and archaea that helps to sustain growth in syntrophic methanogenic communities is reviewed and the process of reverse electron transfer is described, which is a key requirement in obligately syntrophic interactions.
Abstract: Interspecies electron transfer is a key process in methanogenic and sulphate-reducing environments. Bacteria and archaea that live in syntrophic communities take advantage of the metabolic abilities of their syntrophic partner to overcome energy barriers and break down compounds that they cannot digest by themselves. Here, we review the transfer of hydrogen and formate between bacteria and archaea that helps to sustain growth in syntrophic methanogenic communities. We also describe the process of reverse electron transfer, which is a key requirement in obligately syntrophic interactions. Anaerobic methane oxidation coupled to sulphate reduction is also carried out by syntrophic communities of bacteria and archaea but, as we discuss, the exact mechanism of this syntrophic interaction is not yet understood.
1,052 citations
"Methane production from protozoan e..." refers background in this paper
...These include not only the various forms of interspecies electron transfer between bacteria and methanogens (Stams and Plugge, 2009; Malvankar and Lovley, 2014; Rotaru et al., 2014), but also the symbiotic association of protozoa and endosymbiotic methanogens (van Hoek et al., 2000; Fenchel and…...
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