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

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 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 Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community

2004 ◽  
Vol 70 (1) ◽  
pp. 588-596 ◽  
Author(s):  
Maneesha P. Ginige ◽  
Philip Hugenholtz ◽  
Holger Daims ◽  
Michael Wagner ◽  
Jürg Keller ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Microbial Community ◽  
Stable Isotope ◽  
Fluorescence In Situ Hybridization ◽  
Clone Library ◽  
Stable Isotope Probing ◽  
Denitrification Rate ◽  
Biological Nutrient Removal ◽  
Group A

ABSTRACT A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3 −-N mg of mixed-liquor volatile suspended solids (MLVSS)−1 h−1 to a steady-state value of 0.06 mg of NO3 −-N mg of MLVSS−1 h−1 over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [13C]methanol to biomark the DNA of the denitrifiers. The extracted [13C]DNA and [12C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [13C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [12C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [14C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.

scholarly journals Seafloor incubation experiment with deep-sea hydrothermal vent fluid reveals effect of pressure and lag time on autotrophic microbial communities

2021 ◽  
Author(s):  
Caroline S. Fortunato ◽  
David A. Butterfield ◽  
Benjamin Larson ◽  
Noah Lawrence-Slavas ◽  
Christopher K. Algar ◽  
...  
Keyword(s):  
Thermal Stress ◽  
Stable Isotope ◽  
Microbial Communities ◽  
Deep Sea ◽  
Stress Proteins ◽  
Natural Habitat ◽  
Deep Ocean ◽  
Stable Isotope Probing ◽  
Sample Processing

Depressurization and sample processing delays may impact the outcome of shipboard microbial incubations of samples collected from the deep sea. To address this knowledge gap, we developed an ROV-powered incubator instrument to carry out and compare results from in situ and shipboard RNA Stable Isotope Probing (RNA-SIP) experiments to identify the key chemolithoautotrophic microbes and metabolisms in diffuse, low-temperature venting fluids from Axial Seamount. All the incubations showed microbial uptake of labeled bicarbonate primarily by thermophilic autotrophic Epsilonbacteraeota that oxidized hydrogen coupled with nitrate reduction. However, the in situ seafloor incubations showed higher abundances of transcripts annotated for aerobic processes suggesting that oxygen was lost from the hydrothermal fluid samples prior to shipboard analysis. Furthermore, transcripts for thermal stress proteins such as heat shock chaperones and proteases were significantly more abundant in the shipboard incubations suggesting that depressurization induced thermal stress in the metabolically active microbes in these incubations. Together, results indicate that while the autotrophic microbial communities in the shipboard and seafloor experiments behaved similarly, there were distinct differences that provide new insight into the activities of natural microbial assemblages under near-native conditions in the ocean. Importance: Diverse microbial communities drive biogeochemical cycles in Earth’s ocean, yet studying these organisms and processes is often limited by technological capabilities, especially in the deep ocean. In this study, we used a novel marine microbial incubator instrument capable of in situ experimentation to investigate microbial primary producers at deep-sea hydrothermal vents. We carried out identical stable isotope probing experiments coupled to RNA sequencing both on the seafloor and on the ship to examine thermophilic, microbial autotrophs in venting fluids from an active submarine volcano. Our results indicate that microbial communities were significantly impacted by the effects of depressurization and sample processing delay, with shipboard microbial communities more stressed compared to seafloor incubations. Differences in metabolism were also apparent and are likely linked to the chemistry of the fluid at the beginning of the experiment. Microbial experimentation in the natural habitat provides new insights into understanding microbial activities in the ocean.

scholarly journals Stable Isotope Probing for Microbial Iron Reduction in Chocolate Pots Hot Spring, Yellowstone National Park

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Nathaniel W. Fortney ◽  
Shaomei He ◽  
Ajinkya Kulkarni ◽  
Michael W. Friedrich ◽  
Charlotte Holz ◽  
...  
Keyword(s):  
Microbial Community ◽  
Stable Isotope ◽  
Yellowstone National Park ◽  
National Park ◽  
Hot Spring ◽  
Redox Cycling ◽  
Stable Isotope Probing ◽  
Content Type ◽  
Insight Into

ABSTRACTChocolate Pots hot springs (CP) is a circumneutral-pH Fe-rich geothermal feature located in Yellowstone National Park. Previous Fe(III)-reducing enrichment culture studies with CP sediments identified close relatives of known dissimilatory Fe(III)-reducing bacterial (FeRB) taxa, includingGeobacterandMelioribacter. However, the abundances and activities of such organisms in the native microbial community are unknown. Here, we used stable isotope probing experiments combined with 16S rRNA gene amplicon and shotgun metagenomic sequencing to gain an understanding of thein situFe(III)-reducing microbial community at CP. Fe-Si oxide precipitates collected near the hot spring vent were incubated with unlabeled and13C-labeled acetate to target active FeRB. We searched reconstructed genomes for homologs of genes involved in known extracellular electron transfer (EET) systems to identify the taxa involved in Fe redox transformations. Known FeRB taxa containing putative EET systems (Geobacter,Ignavibacteria) increased in abundance under acetate-amended conditions, whereas genomes related toIgnavibacteriumandThermodesulfovibriothat contained putative EET systems were recovered from incubations without electron donor. Our results suggest that FeRB play an active role in Fe redox cycling within Fe-Si oxide-rich deposits located at the hot spring vent.IMPORTANCEThe identification of past near-surface hydrothermal environments on Mars emphasizes the importance of using modern Earth environments, such as CP, to gain insight into potential Fe-based microbial life on other rocky worlds, as well as ancient Fe-rich Earth ecosystems. By combining stable carbon isotope probing techniques and DNA sequencing technology, we gained insight into the pathways of microbial Fe redox cycling at CP. The results suggest that microbial Fe(III) oxide reduction is prominentin situ, with important implications for the generation of geochemical and stable Fe isotopic signatures of microbial Fe redox metabolism within Fe-rich circumneutral-pH thermal spring environments on Earth and Mars.

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