scholarly journals Elucidating Syntrophic Butyrate-Degrading Populations in Anaerobic Digesters Using Stable-Isotope-Informed Genome-Resolved Metagenomics

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Ryan M. Ziels ◽  
Masaru K. Nobu ◽  
Diana Z. Sousa
Keyword(s):  
Electron Transfer ◽  
Stable Isotope ◽  
Stable Isotope Probing ◽  
Microbial Interactions ◽  
Metabolic Reconstruction ◽  
Metabolic Potential ◽  
Metabolic Cooperation ◽  
Anaerobic Digesters ◽  
Slow Growing

ABSTRACT Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable-isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in 13C after growing on [13C]butyrate. Differential abundance analysis of recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in heavy [13C]DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (<95%) with any cultured representative species, indicating that it is a novel species that plays a significant role in syntrophic butyrate degradation within anaerobic digesters. The Methanothrix genome contained the complete pathway for acetoclastic methanogenesis, indicating that it was enriched in 13C from syntrophic acetate transfer. This study demonstrates the potential of stable-isotope-informed genome-resolved metagenomics to identify in situ interspecies metabolic cooperation within syntrophic consortia important to anaerobic waste treatment as well as global carbon cycling. IMPORTANCE Predicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in situ metabolic activities of uncultured species may enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in situ function of mixed microbiomes by combining stable-isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C4 fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve “who is doing what” by obtaining confirmatory assimilation of the labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes.

scholarly journals Elucidating syntrophic butyrate-degrading populations in anaerobic digesters using stable isotope-informed genome-resolved metagenomics

2019 ◽  
Author(s):  
Ryan M. Ziels ◽  
Masaru K. Nobu ◽  
Diana Z. Sousa
Keyword(s):  
Electron Transfer ◽  
Stable Isotope ◽  
Stable Isotope Probing ◽  
Microbial Interactions ◽  
Metabolic Reconstruction ◽  
Metabolic Potential ◽  
Metabolic Cooperation ◽  
Anaerobic Digesters ◽  
Slow Growing

AbstractLinking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in 13C after feeding 13C-labeled butyrate. Differential abundance analysis on recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in the heavy 13C DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas, and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (< 95%) with any cultured representative species, indicating it is a novel species that plays a significant role in syntrophic butyrate degradation within anaerobic digesters. The Methanothrix genome contained the complete pathway for aceticlastic methanogenesis, indicating that it was enriched in 13C from syntrophic acetate transfer. This study demonstrates the potential of stable-isotope-informed genome-resolved metagenomics to elucidate the nature of metabolic cooperation in slow-growing uncultured microbial populations, such as syntrophic bacteria and methanogens, that are important to waste treatment as well as global carbon cycling.ImportancePredicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in-situ metabolic activities of uncultured species could enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in-situ function of mixed microbiomes by combining DNA-stable isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C4 fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve ‘who is doing what’ by obtaining confirmatory assimilation of labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes.

scholarly journals Subgroup level differences of physiological activities in marine Lokiarchaeota

2020 ◽  
Author(s):  
Xiuran Yin ◽  
Mingwei Cai ◽  
Yang Liu ◽  
Guowei Zhou ◽  
Tim Richter-Heitmann ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Marine Sediments ◽  
Inorganic Carbon ◽  
Bacterial Biomass ◽  
Stable Isotope Probing ◽  
Aromatic Compound Degradation ◽  
Lactate Degradation ◽  
Globally Distributed ◽  
Mud Area

Abstract Asgard is a recently discovered archaeal superphylum, closely linked to the emergence of eukaryotes. Among Asgard archaea, Lokiarchaeota are abundant in marine sediments, but their in situ activities are largely unknown except for Candidatus ‘Prometheoarchaeum syntrophicum’. Here, we tracked the activity of Lokiarchaeota in incubations with Helgoland mud area sediments (North Sea) by stable isotope probing (SIP) with organic polymers, 13C-labelled inorganic carbon, fermentation intermediates and proteins. Within the active archaea, we detected members of the Lokiarchaeota class Loki-3, which appeared to mixotrophically participate in the degradation of lignin and humic acids while assimilating CO2, or heterotrophically used lactate. In contrast, members of the Lokiarchaeota class Loki-2 utilized protein and inorganic carbon, and degraded bacterial biomass formed in incubations. Metagenomic analysis revealed pathways for lactate degradation, and involvement in aromatic compound degradation in Loki-3, while the less globally distributed Loki-2 instead rely on protein degradation. We conclude that Lokiarchaeotal subgroups vary in their metabolic capabilities despite overlaps in their genomic equipment, and suggest that these subgroups occupy different ecologic niches in marine sediments.

scholarly journals Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography

2005 ◽  
Vol 71 (12) ◽  
pp. 8683-8691 ◽  
Author(s):  
Maneesha P. Ginige ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
In Situ Hybridization ◽  
Stable Isotope ◽  
Activated Sludge ◽  
Fluorescent In Situ Hybridization ◽  
Batch Reactor ◽  
External Source ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
The Impact

ABSTRACT The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.

scholarly journals Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow Growing Methane Based Microbial Communities

2016 ◽  
Vol 7 ◽  
Author(s):  
Jeffrey J. Marlow ◽  
Connor T. Skennerton ◽  
Zhou Li ◽  
Karuna Chourey ◽  
Robert L. Hettich ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Microbial Communities ◽  
Stable Isotope Probing ◽  
Slow Growing

scholarly journals Methyl-compound use and slow growth characterize microbial life in 2-km-deep subseafloor coal and shale beds

2017 ◽  
Vol 114 (44) ◽  
pp. E9206-E9215 ◽  
Author(s):  
Elizabeth Trembath-Reichert ◽  
Yuki Morono ◽  
Akira Ijiri ◽  
Tatsuhiko Hoshino ◽  
Katherine S. Dawson ◽  
...  
Keyword(s):  
Stable Isotope Probing ◽  
Coal Bed ◽  
Rrna Gene ◽  
Deuterated Water ◽  
Diverse Range ◽  
Microbial Life ◽  
Ocean Drilling ◽  
Slow Growing ◽  
Deep Subseafloor

The past decade of scientific ocean drilling has revealed seemingly ubiquitous, slow-growing microbial life within a range of deep biosphere habitats. Integrated Ocean Drilling Program Expedition 337 expanded these studies by successfully coring Miocene-aged coal beds 2 km below the seafloor hypothesized to be “hot spots” for microbial life. To characterize the activity of coal-associated microorganisms from this site, a series of stable isotope probing (SIP) experiments were conducted using intact pieces of coal and overlying shale incubated at in situ temperatures (45 °C). The 30-month SIP incubations were amended with deuterated water as a passive tracer for growth and different combinations of13C- or15N-labeled methanol, methylamine, and ammonium added at low (micromolar) concentrations to investigate methylotrophy in the deep subseafloor biosphere. Although the cell densities were low (50–2,000 cells per cubic centimeter), bulk geochemical measurements and single-cell–targeted nanometer-scale secondary ion mass spectrometry demonstrated active metabolism of methylated substrates by the thermally adapted microbial assemblage, with differing substrate utilization profiles between coal and shale incubations. The conversion of labeled methylamine and methanol was predominantly through heterotrophic processes, with only minor stimulation of methanogenesis. These findings were consistent with in situ and incubation 16S rRNA gene surveys. Microbial growth estimates in the incubations ranged from several months to over 100 y, representing some of the slowest direct measurements of environmental microbial biosynthesis rates. Collectively, these data highlight a small, but viable, deep coal bed biosphere characterized by extremely slow-growing heterotrophs that can utilize a diverse range of carbon and nitrogen substrates.

scholarly journals Identification of benzene-degrading Proteobacteria in a constructed wetland by employing in situ microcosms and RNA-stable isotope probing

2019 ◽  
Vol 104 (4) ◽  
pp. 1809-1820 ◽  
Author(s):  
Henrike Nitz ◽  
Márcia Duarte ◽  
Ruy Jauregui ◽  
Dietmar H. Pieper ◽  
Jochen A. Müller ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Constructed Wetland ◽  
Stable Isotope Probing ◽  
In Situ Microcosms
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