Microbial community ecology of marine methane seeps

The detailed investigation of microbial communities, e.g. of soil or hydrothermal vent ecosystems, greatly improved our understanding of the diversity, habitat preferences and functions of microorganisms and their impact on global element cycles. The aim of this thesis was a detailed analysis of the diversity, abundance and distribution of micoorganisms at marine methane seeps and the mechanisms that govern community assembly at these sites. The seep ecosystems were investigated using geochemical analyses, gene libraries, pyrosequencing, community fingerprinting and fluorescence in situ hybridization. Cold seep ecosystems hosted distinct microbial communities that differed from those of the surrounding seabed and were unique microbial habitat patches in the deep sea. The communities also greatly differed between seeps, covered broad ranges of richness and evenness and showed high degrees of endemism. However, despite the differences all seeps were inhabited by certain organisms the cold seep microbiome - including key functional clades of anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria. Additionally, aerobic methanotrophs and thiotrophs were found at all seeps where oxygen was present. These key functional clades seemed to be influenced by environmental parameters, such as temperature, fluid flux, sediment depth and faunal activity. Bioirrigation by ampharetid tubeworms, for instance, created a habitat for aerobic Methylococcales, whereas vesicomyid clams seemed to favor the establishment of the clade ANME-2c. Thus, niche-based processes played an important role for the community assembly at seep ecosystems. However, most of the seeps seemed to be clearly dominated by a few, globally distributed operational taxonomic units at 97% 16S rRNA gene identity (OTU0.03) of each key functional clade. Some of these OTU0.03 were rare at some seep ecosystems and abundant at others. Moreover, some findings suggested that rare organisms became abundant because the environmental conditions at the seep changed supporting the importance of species sorting at seep communities. Finally, the succession of microbial communities and the emergence of ecosystem function at a cold seep were monitored showing that it may take years to develop fully functioning communities that efficiently remove the potential greenhouse gas methane. Overall this work may help to resolve the mysteries of microbial community ecology at cold seep ecosystems

Hydrocarbon seepage in the deep seabed links subsurface and seafloor biospheres

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Chakraborty, A., Ruff, S. E., Dong, X., Ellefson, E. D., Li, C., Brooks, J. M., McBee, J., Bernard, B. B., & Hubert, C. R. J. Hydrocarbon seepage in the deep seabed links subsurface and seafloor biospheres. Proceedings of the National Academy of Sciences of the United States of America, 117(20), (2020): 11029-11037, doi: 10.1073/pnas.2002289117.Marine cold seeps transmit fluids between the subseafloor and seafloor biospheres through upward migration of hydrocarbons that originate in deep sediment layers. It remains unclear how geofluids influence the composition of the seabed microbiome and if they transport deep subsurface life up to the surface. Here we analyzed 172 marine surficial sediments from the deep-water Eastern Gulf of Mexico to assess whether hydrocarbon fluid migration is a mechanism for upward microbial dispersal. While 132 of these sediments contained migrated liquid hydrocarbons, evidence of continuous advective transport of thermogenic alkane gases was observed in 11 sediments. Gas seeps harbored distinct microbial communities featuring bacteria and archaea that are well-known inhabitants of deep biosphere sediments. Specifically, 25 distinct sequence variants within the uncultivated bacterial phyla Atribacteria and Aminicenantes and the archaeal order Thermoprofundales occurred in significantly greater relative sequence abundance along with well-known seep-colonizing members of the bacterial genus Sulfurovum, in the gas-positive sediments. Metabolic predictions guided by metagenome-assembled genomes suggested these organisms are anaerobic heterotrophs capable of nonrespiratory breakdown of organic matter, likely enabling them to inhabit energy-limited deep subseafloor ecosystems. These results point to petroleum geofluids as a vector for the advection-assisted upward dispersal of deep biosphere microbes from subsurface to surface environments, shaping the microbiome of cold seep sediments and providing a general mechanism for the maintenance of microbial diversity in the deep sea.We wish to thank Jody Sandel as well as the crew of R/V GeoExplorer for collection of piston cores, onboard core processing, sample preservation, and shipment. Cynthia Kwan and Oliver Horanszky are thanked for assistance with amplicon library preparation. We also wish to thank Jayne Rattray, Daniel Gittins, and Marc Strous for valuable discussions and suggestions, and Rhonda Clark for research support. Collaborations with Andy Mort from the Geological Survey of Canada, and Richard Hatton from Geoscience Wales are also gratefully acknowledged. This work was financially supported by a Mitacs Elevate Postdoctoral Fellowship awarded to A.C.; an Alberta Innovates-Technology Futures/Eyes High Postdoctoral Fellowship to S.E.R.; and a Natural Sciences and Engineering Research Council Strategic Project Grant, a Genome Canada Genomics Applications Partnership Program grant, a Canada Foundation for Innovation grant (CFI-JELF 33752) for instrumentation, and Campus Alberta Innovates Program Chair funding to C.R.J.H

Methane oxidation and methylotroph population dynamics in groundwater mesocosms

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Kuloyo, O., Ruff, S. E., Cahill, A., Connors, L., Zorz, J. K., de Angelis, I. H., Nightingale, M., Mayer, B., & Strous, M. Methane oxidation and methylotroph population dynamics in groundwater mesocosms. Environmental Microbiology. (2020), doi:10.1111/1462-2920.14929.Extraction of natural gas from unconventional hydrocarbon reservoirs by hydraulic fracturing raises concerns about methane migration into groundwater. Microbial methane oxidation can be a significant methane sink. Here, we inoculated replicated, sand‐packed, continuous mesocosms with groundwater from a field methane release experiment. The mesocosms experienced thirty‐five weeks of dynamic methane, oxygen and nitrate concentrations. We determined concentrations and stable isotope signatures of methane, carbon dioxide and nitrate and monitored microbial community composition of suspended and attached biomass. Methane oxidation was strictly dependent on oxygen availability and led to enrichment of 13C in residual methane. Nitrate did not enhance methane oxidation under oxygen limitation. Methylotrophs persisted for weeks in the absence of methane, making them a powerful marker for active as well as past methane leaks. Thirty‐nine distinct populations of methylotrophic bacteria were observed. Methylotrophs mainly occurred attached to sediment particles. Abundances of methanotrophs and other methylotrophs were roughly similar across all samples, pointing at transfer of metabolites from the former to the latter. Two populations of Gracilibacteria (Candidate Phyla Radiation) displayed successive blooms, potentially triggered by a period of methane famine. This study will guide interpretation of future field studies and provides increased understanding of methylotroph ecophysiology.The authors acknowledge funding from the Alberta Innovates Technology Futures (AITF), and University of Calgary Eyes High Doctoral Scholarships (O.O.K., J.K.Z.) and AITF/Eyes High Postdoctoral Fellowships (S.E.R.), as well as the PROMOS Internship Abroad Scholarship by the German Academic Exchange Service (I.H.d.A.). Additional support was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC), Strategic Project Grant no. 463045‐14, the Campus Alberta Innovation Chair Program (M.S.), Alberta Innovates, The Canadian Foundation for Innovation (M.S.), the Alberta Small Equipment Grant Program (M.S.) and an NSERC Discovery Grant (M.S. and B.M.)

Degradation of biological macromolecules supports uncultured microbial populations in Guaymas Basin hydrothermal sediments

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Castro, S. P., Borton, M. A., Regan, K., de Angelis, I. H., Wrighton, K. C., Teske, A. P., Strous, M., & Ruff, S. E. Degradation of biological macromolecules supports uncultured microbial populations in Guaymas Basin hydrothermal sediments. Isme Journal. (2021), https://doi.org/10.1038/s41396-021-01026-5.Hydrothermal sediments contain large numbers of uncultured heterotrophic microbial lineages. Here, we amended Guaymas Basin sediments with proteins, polysaccharides, nucleic acids or lipids under different redox conditions and cultivated heterotrophic thermophiles with the genomic potential for macromolecule degradation. We reconstructed 20 metagenome-assembled genomes (MAGs) of uncultured lineages affiliating with known archaeal and bacterial phyla, including endospore-forming Bacilli and candidate phylum Marinisomatota. One Marinisomatota MAG had 35 different glycoside hydrolases often in multiple copies, seven extracellular CAZymes, six polysaccharide lyases, and multiple sugar transporters. This population has the potential to degrade a broad spectrum of polysaccharides including chitin, cellulose, pectin, alginate, chondroitin, and carrageenan. We also describe thermophiles affiliating with the genera Thermosyntropha, Thermovirga, and Kosmotoga with the capability to make a living on nucleic acids, lipids, or multiple macromolecule classes, respectively. Several populations seemed to lack extracellular enzyme machinery and thus likely scavenged oligo- or monomers (e.g., MAGs affiliating with Archaeoglobus) or metabolic products like hydrogen (e.g., MAGs affiliating with Thermodesulfobacterium or Desulforudaceae). The growth of methanogens or the production of methane was not observed in any condition, indicating that the tested macromolecules are not degraded into substrates for methanogenesis in hydrothermal sediments. We provide new insights into the niches, and genomes of microorganisms that actively degrade abundant necromass macromolecules under oxic, sulfate-reducing, and fermentative thermophilic conditions. These findings improve our understanding of the carbon flow across trophic levels and indicate how primary produced biomass sustains complex and productive ecosystems.We are grateful to the captain and crew of the R/V Atlantis AT37-06 as well as the crew of the human occupied vehicle Alvin for their tireless support. Sampling at Guaymas Basin was supported by NSF (OCE-1357238)

Freezing Tolerance of Thermophilic Bacterial Endospores in Marine Sediments

Dormant endospores of anaerobic, thermophilic bacteria found in cold marine sediments offer a useful model for studying microbial biogeography, dispersal, and survival. The dormant endospore phenotype confers resistance to unfavorable environmental conditions, allowing dispersal to be isolated and studied independently of other factors such as environmental selection. To study the resilience of thermospores to conditions relevant for survival in extreme cold conditions, their viability following different freezing treatments was tested. Marine sediment was frozen at either −80°C or −20°C for 10 days prior to pasteurization and incubation at +50°C for 21 days to assess thermospore viability. Sulfate reduction commenced at +50°C following both freezing pretreatments indicating persistence of thermophilic endospores of sulfate-reducing bacteria. The onset of sulfate reduction at +50°C was delayed in −80°C pretreated microcosms, which exhibited more variability between triplicates, compared to −20°C pretreated microcosms and parallel controls that were not frozen in advance. Microbial communities were evaluated by 16S rRNA gene amplicon sequencing, revealing an increase in the relative sequence abundance of thermophilic endospore-forming Firmicutes in all microcosms. Different freezing pretreatments (−80°C and −20°C) did not appreciably influence the shift in overall bacterial community composition that occurred during the +50°C incubations. Communities that had been frozen prior to +50°C incubation showed an increase in the relative sequence abundance of operational taxonomic units (OTUs) affiliated with the class Bacilli, relative to unfrozen controls. These results show that freezing impacts but does not obliterate thermospore populations and their ability to germinate and grow under appropriate conditions. Indeed the majority of the thermospore OTUs detected in this study (21 of 22) could be observed following one or both freezing treatments. These results are important for assessing thermospore viability in frozen samples and following cold exposure such as the very low temperatures that would be encountered during panspermia

Microbial communities under distinct thermal and geochemical regimes in axial and off-axis sediments of Guaymas Basin

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Teske, A., Wegener, G., Chanton, J. P., White, D., MacGregor, B., Hoer, D., de Beer, D., Zhuang, G., Saxton, M. A., Joye, S. B., Lizarralde, D., Soule, S. A., & Ruff, S. E. Microbial communities under distinct thermal and geochemical regimes in axial and off-axis sediments of Guaymas Basin. Frontiers in Microbiology, 12, (2021): 633649, https://doi.org/10.3389/fmicb.2021.633649.Cold seeps and hydrothermal vents are seafloor habitats fueled by subsurface energy sources. Both habitat types coexist in Guaymas Basin in the Gulf of California, providing an opportunity to compare microbial communities with distinct physiologies adapted to different thermal regimes. Hydrothermally active sites in the southern Guaymas Basin axial valley, and cold seep sites at Octopus Mound, a carbonate mound with abundant methanotrophic cold seep fauna at the Central Seep location on the northern off-axis flanking regions, show consistent geochemical and microbial differences between hot, temperate, cold seep, and background sites. The changing microbial actors include autotrophic and heterotrophic bacterial and archaeal lineages that catalyze sulfur, nitrogen, and methane cycling, organic matter degradation, and hydrocarbon oxidation. Thermal, biogeochemical, and microbiological characteristics of the sampling locations indicate that sediment thermal regime and seep-derived or hydrothermal energy sources structure the microbial communities at the sediment surface.Research on Guaymas Basin in the Teske lab is supported by NSF Molecular and cellular Biology grant 1817381 “Collaborative Research: Next generation physiology: a systems-level understanding of microbes driving carbon cycling in marine sediments”. Sampling in Guaymas Basin was supported by collaborative NSF Biological Oceanography grants 1357238 and 1357360 “Collaborative Research: Microbial carbon cycling and its interaction with sulfur and nitrogen transformations in Guaymas Basin hydrothermal sediments” to AT and SJ, respectively. SER was supported by an AITF/Eyes High Postdoctoral Fellowship and start-up funds provided by the Marine Biological Laboratory

Microbial Communities in Methane- and Short Chain Alkane-Rich Hydrothermal Sediments of Guaymas Basin

The hydrothermal sediments of Guaymas Basin, an active spreading center in the Gulf of California (Mexico), are rich in porewater methane, short-chain alkanes, sulfate and sulfide, and provide a model system to explore habitat preferences of microorganisms, including sulfate-dependent, methane- and short chain alkane-oxidizing microbial communities. In this study, hot sediments (above 60°C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed “Mat Mound”) were characterized by porewater geochemistry of methane, C2–C6 short-chain alkanes, sulfate, sulfide, sulfate reduction rate measurements, in situ temperature gradients, bacterial and archaeal 16S rRNA gene clone libraries and V6 tag pyrosequencing. The most abundantly detected groups in the Mat mound sediments include anaerobic methane-oxidizing archaea of the ANME-1 lineage and its sister clade ANME-1Guaymas, the uncultured bacterial groups SEEP-SRB2 within the Deltaproteobacteria and the separately branching HotSeep-1 Group; these uncultured bacteria are candidates for sulfate-reducing alkane oxidation and for sulfate-reducing syntrophy with ANME archaea. The archaeal dataset indicates distinct habitat preferences for ANME-1, ANME-1-Guaymas, and ANME-2 archaea in Guaymas Basin hydrothermal sediments. The bacterial groups SEEP-SRB2 and HotSeep-1 co-occur with ANME-1 and ANME-1Guaymas in hydrothermally active sediments underneath microbial mats in Guaymas Basin. We propose the working hypothesis that this mixed bacterial and archaeal community catalyzes the oxidation of both methane and short-chain alkanes, and constitutes a microbial community signature that is characteristic for hydrothermal and/or cold seep sediments containing both substrates

The microbial biodiversity at the archeological site of Tel Megiddo (Israel)

IntroductionThe ancient city of Tel Megiddo in the Jezreel Valley (Israel), which lasted from the Neolithic to the Iron Age, has been continuously excavated since 1903 and is now recognized as a World Heritage Site. The site features multiple ruins in various areas, including temples and stables, alongside modern constructions, and public access is allowed in designated areas. The site has been studied extensively since the last century; however, its microbiome has never been studied. We carried out the first survey of the microbiomes in Tel Megiddo. Our objectives were to study (i) the unique microbial community structure of the site, (ii) the variation in the microbial communities across areas, (iii) the similarity of the microbiomes to urban and archeological microbes, (iv) the presence and abundance of potential bio-corroding microbes, and (v) the presence and abundance of potentially pathogenic microbes.MethodsWe collected 40 swab samples from ten major areas and identified microbial taxa using next-generation sequencing of microbial genomes. These genomes were annotated and classified taxonomically and pathogenetically.ResultsWe found that eight phyla, six of which exist in all ten areas, dominated the site (>99%). The relative sequence abundance of taxa varied between the ruins and the sampled materials and was assessed using all metagenomic reads mapping to a respective taxon. The site hosted unique taxa characteristic of the built environment and exhibited high similarity to the microbiome of other monuments. We identified acid-producing bacteria that may pose a risk to the site through biocorrosion and staining and thus pose a danger to the site’s preservation. Differences in the microbiomes of the publicly accessible or inaccessible areas were insignificant; however, pathogens were more abundant in the former.DiscussionWe found that Tel Megiddo combines microbiomes of arid regions and monuments with human pathogens. The findings shed light on the microbial community structures and have relevance for bio-conservation efforts and visitor health

Microbial cell counts from Canadian Prairie groundwater ecosystems

Groundwater ecosystems are globally widespread yet still poorly understood. We therefore investigated the total microbial cell counts of 78 groundwater samples from aquifers in the Canadian Prairie, collected between 2015 and 2020. The study area comprised 14 major aquifers and a geographic area of ~210.000 km2. The goal of the study was to understand the links between the biogeochemistry and microbial ecology of groundwater ecosystems in diverse geological settings on a broad spatial scale. Details concerning microscopy and cell enumeration can be found in the associated publication by Ruff et al. 2023