New insights into microbial ecology through subtle nucleotide variation

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 7 (2016): 1318, doi:10.3389/fmicb.2016.01318.Characterizing the community structure of naturally occurring microbes through marker gene amplicons has gained widespread acceptance for profiling microbial populations. The 16S ribosomal RNA (rRNA) gene provides a suitable target for most studies since (1) it meets the criteria for robust markers of evolution, e.g., both conserved and rapidly evolving regions that do not undergo horizontal gene transfer, (2) microbial ecologists have identified widely adopted primers and protocols for generating amplicons for sequencing, (3) analyses of both cultivars and environmental DNA have generated well-curated databases for taxonomic profiling, and (4) bioinformaticians and computational biologists have published comprehensive software tools for interpreting the data and generating publication-ready figures. Since the initial descriptions of high-throughput sequencing of 16S rRNA gene amplicons to survey microbial diversity, we have witnessed an explosion of association-based inferences of interactions between microbes and their environment.AME was supported by the University of Chicago and the Marine Biological Laboratory collaboration award

The Pen Duick Escarpment off Morocco: A promising biogeochemically active carbonate mound laboratory (MiCROSYSTEMS)

Carbonate mud mounds, found in marine environments from shallow- to deep-water settings, span from Proterozoic to recent times. During the past decades, numerous active venting fields were discovered in deep marine environments and became a subject of extensive study for marine scientists. Mound building seems to be a fundamental but still enigmatic strategy for life. Various arguments suggest that microorganisms are playing a major role in the reef development, mound formation and biodiversity. Therefore, it is important to evaluate the microbial mediated processes of carbonate precipitation.Cold-water coral reefs thriving on carbonate mounds were discovered in the late 1990’s off western Ireland. An exploratory cruise of RV Belgica in 2002 off Morocco has led to the discovery of apparently juvenile mounds in water depths of 500-600 m, topping a cliff - the Pen Duick escarpment - flanked by giant mud volcanoes. Subsequent cruises have confirmed the colonization by deep-water corals and have unveiled extensive fields of seep-related carbonate crusts in the off-reef regions. Long cores taken in 2004 indicate that the ‘Pen Duick’ mounds, in which microbial action was demonstrated by a strong emission of hydrogen sulphide, may be considered as giant biogeochemical reactors. The mound sediments were dated 2 kyrs B.P. at the surface and 20 kyrs B.P. in a depth of 6 mbsf.A 450 cm long gravity core, coming from one of these juvenile mounds, was sampled and analyzed for mineralogy, stable isotopes composition, geochemistry, and microbial communities. Most of the sediment consists of calcite (coccoliths), quartz and dolomite. At a depth of 4 mbsf, we found hardened nodule-like structures, embedded in grey mud containing cold-water coral pieces. The presence of 20-30% of dolomite, in the carbonate phase, suggests a microbial influence during mineral formation. Preliminary results of the pore water geochemistry indicate a reactive sulphate – methane interface at 3.8 mbsf. In this layer we focused our studies on the microbial communities, such as methanogens, methanotrophs and sulphate reducers. The trend of the d13C values in digenetic carbonate supports the assumption of microbial activity in this section of the core. In order to define the primary microbial community involved in carbonate precipitation, we did direct culturing, DNA isolation and PCR analysis of three functional genes, the a subunit (mcrA) of the methyl-coenzyme M reductase (MCR), the a subunit (pmoA) of the particulate methane monooxygenase (MMO) and the a and ß subunits (dsrA and dsrB, respectively) of the dissimilatory sulfite reductase (DSR). These enzymes are involved in methanogenesis, methanotrophy and sulphate reduction biochemical pathways, respectively. In summary, our initial results demonstrate that the Pen Duick carbonate mound can be considered as a natural laboratory in which to study cold-water coral ecosystems associated with microbial activity

Simulation of the deep-sea biosphere by a continuous high-pressure bioreactor

In ocean system Anaerobic Oxidation of Methane (AOM) followed by carbonate precipitation has a significant effect on the climate regulation, since this process avoids large methane emissions to the atmosphere and fixes carbon dioxide into carbonate structures. However the main difficulty to study AOM is that the consortia involved have extremely long doubling time (2-7 months) at ambient or low pressures. To simulate the in situ condition better and obtain a faster growth, we designed and constructed a unique continuous high-pressure bioreactor. The reactor can reach pressure up to 100 bars, representing a depth of 1000m below sea level; it can be operated in continuous or non-continuous style, simulating the different types of methane resource. By the help of this high pressure bioreactor system, we are also able to study the effect of environmental factors on AOM activity and on microbial community. Captain Arutyunov Mud Volcano (Gulf of Cadiz) sediment has been used as biomass resource and different molecular techniques (DGGE, cloning library, FISH) have been applied to examine the microbial community structure. By increasing methane partial pressure, an immediate increase of AOM activity has been observed before significant enrichment of biomass. A continuous methane flux is necessary to obtain optimal AOMactivity. Bacterial community is more sensitive to the change of pressure compared with archaeal community

Ecological succession and stochastic variation in the assembly of Arabidopsis thaliana phyllosphere communities

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in mBio 5 (2014): e00682-13, doi:10.1128/mBio.00682-13.Bacteria living on the aerial parts of plants (the phyllosphere) are globally abundant and ecologically significant communities and can have significant effects on their plant hosts. Despite their importance, little is known about the ecological processes that drive phyllosphere dynamics. Here, we describe the development of phyllosphere bacterial communities over time on the model plant Arabidopsis thaliana in a controlled greenhouse environment. We used a large number of replicate plants to identify repeatable dynamics in phyllosphere community assembly and reconstructed assembly history by measuring the composition of the airborne community immigrating to plant leaves. We used more than 260,000 sequences from the v5v6 hypervariable region of the 16S rRNA gene to characterize bacterial community structure on 32 plant and 21 air samples over 73 days. We observed strong, reproducible successional dynamics: phyllosphere communities initially mirrored airborne communities and subsequently converged to a distinct community composition. While the presence or absence of particular taxa in the phyllosphere was conserved across replicates, suggesting strong selection for community composition, the relative abundance of these taxa was highly variable and related to the spatial association of individual plants. Our results suggest that stochastic events in early colonization, coupled with dispersal limitation, generated alternate trajectories of bacterial community assembly within the context of deterministic selection for community membership.Funding was provided by the J. Unger Vetleson Foundation to S.L.S

Biogeochemistry of carbonate mounds from the Pen Duick escarpment in the Gulf of Cadiz

In the Gulf of Cadiz, carbonate mounds build by cold-water corals were recently discovered on the Renard Ridge, a zone of active fluid flow and mud volcanism. Their sizes vary from 25 to more than 60 m high, at a depth of 520 m and they are aligned along the ridge axis. These mounds, located in the close vicinity of fluid flow markers such as carbonate crusts and mud volcanoes, provided a novel opportunity to study a possible fluid flow control on the mound processes and distribution. Previous geochemical studies on the southernmost mound of the ridge indeed showed that this mound was located on focused fluid flow compared to surrounding sediments, and we observed typical profiles of methane migration and anoxic oxidation (AOM) at 3,8 m below the sea floor within the mound. Such AOM occurrence imprinted a characteristic d13C signature (down to –21,9 %¸ Vs. PDB) and significantly contributes to the overall carbonate budget of the mound.During the recent R/V Maria S. Merian cruise (April-June 2006), we sampled by mean of a gravity corer six new structures likely to be cold-water carbonate mounds, along the Pen Duick escarpment and the Renard Ridge. Our aim was to determine if the geochemical profiles observed in the first mound could be generalized to all the mounds in this area.Each core yielded a full sequence of cold-water corals down to about 5 meters below the sea floor. Hence, the numerous knoll-like structures revealed by high-resolution bathymetry along the ridge are indeed carbonate mounds build by cold-water corals and the entire Ridge has been massively colonized by corals. No live reef-forming coral could be recovered from the cores, nor observed by towed video instruments. Then, fluid migration seems to be a common feature all along the ridge. However, important discrepancies were observed: methane concentrations are higher and sulfate gradients steeper on both side of the ridge, whereas the central part of the ridge seems less active in term of fluid migration. In this case, the sulfate to methane transition zone could not be reached using conventional gravity corer. In order to obtain the full biogeochemical picture of these mounds, the use of a long piston corer, or drilling devices, will be required.The reasons of the formation of massive reefs in this area are still unknown and are probably linked to locally enhanced hydrologic conditions. However, it is possible that cold-water coral could have benefited from the hard substrate and the topographic elevations provided by fluid related structures such as carbonate crusts, chimneys and clasts, as observed in several other locations in the Gulf of Cadiz

Stratified community responses to methane and sulfate supplies in mud volcano deposits: insights from an <i>in vitro</i> experiment

Numerous studies on marine prokaryotic communities have postulated that a process of anaerobic oxidation of methane (AOM) coupled with sulfate reduction (SR) is the main methane sink in the world's oceans. AOM has also been reported in the deep biosphere. But the responses of the primary microbial players in eliciting changes in geochemical environments, specifically in methane and sulfate supplies, have yet to be fully elucidated. Marine mud volcanoes (MVs) expel a complex fluid mixture of which methane is the primary component, forming an environment in which AOM is a common phenomenon. In this context, we attempted to identify how the prokaryotic community would respond to changes in methane and sulfate intensities, which often occur in MV environments in the form of eruptions, diffusions or seepage. We applied an integrated approach, including (i) biochemical surveys of pore water originated from MV, (ii) in vitro incubation of mud breccia, and (iii) prokaryotic community structure analysis. Two distinct AOM regions were clearly detected. One is related to the sulfate methane transition zone (SMTZ) at depth of 30-55 cm below the sea floor (bsf); the second is at 165-205 cm bsf with ten times higher rates of AOM and SR. This finding contrasts with the sulfide concentrations in pore waters and supports the suggestion that potential AOM activity below the SMTZ might be an important methane sink that is largely ignored or underestimated in oceanic methane budget calculations. Moreover, the incubation conditions below the SMTZ favor the growth of methanotrophic archaeal group ANME-2 compared to ANME-1, and promote the rapid growth and high diversity of bacterial communities. These incubation conditions also promote the increase of richness in bacterial communities. Our results provide direct evidence of the mechanisms by which deep AOM processes can affect carbon cycling in the deep biosphere and global methane biochemistry

Oligotyping : differentiating between closely related microbial taxa using 16S rRNA gene data

© The Authors. Methods in Ecology and Evolution © 2013 British Ecological Society.. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Methods in Ecology and Evolution 4 (2013): 1111–1119, doi:10.1111/2041-210X.12114.Bacteria comprise the most diverse domain of life on Earth, where they occupy nearly every possible ecological niche and play key roles in biological and chemical processes. Studying the composition and ecology of bacterial ecosystems and understanding their function are of prime importance. High-throughput sequencing technologies enable nearly comprehensive descriptions of bacterial diversity through 16S ribosomal RNA gene amplicons. Analyses of these communities generally rely upon taxonomic assignments through reference data bases or clustering approaches using de facto sequence similarity thresholds to identify operational taxonomic units. However, these methods often fail to resolve ecologically meaningful differences between closely related organisms in complex microbial data sets. In this paper, we describe oligotyping, a novel supervised computational method that allows researchers to investigate the diversity of closely related but distinct bacterial organisms in final operational taxonomic units identified in environmental data sets through 16S ribosomal RNA gene data by the canonical approaches. Our analysis of two data sets from two different environments demonstrates the capacity of oligotyping at discriminating distinct microbial populations of ecological importance. Oligotyping can resolve the distribution of closely related organisms across environments and unveil previously overlooked ecological patterns for microbial communities. The URL http://oligotyping.org offers an open-source software pipeline for oligotyping.This work was supported by the National Institutes of Health [1UH2DK083993 to M.L.S.] and the Alfred P. Sloan Foundation

Authigenic carbonates from cold-water carbonate mounds in the Gulf of Cadiz: Microbial diversity and imprint on carbonate minerals

The Pen Duick Escarpment off Morocco consists of recent carbonate mounds in water depths of 500-600 m, flanked by giant mud volcanoes. These mounds are covered by mainly lifeless cold-water corals and have been associated with extensive fields of seep-related carbonates in off-reef regions. Three piston cores (from 350 to 640 cm long), coming from different sites on these juvenile mounds, were sampled and analyzed for mineralogy, stable isotopic composition of carbonates, geochemistry, and microbial communities. Most of the sediment comprises pelagic calcite (coccoliths), detrital quartz and authigenic dolomite, often observed encasing coccoliths. The decalcification of the sediment resulted in a dolomite dominated matrix that showed stable carbon istotope values of as low as -30 permil in contrast to the bulk sample values of -7 to -15 permil, which implies the involvement of microbes in the production of bicarbonate ions. Initial results from 16S rRNA gene clone libraries support the theory, that anaerobic oxidation of methane is one of the most important biogeochemical process leading to carbonate precipitation. Preliminary results of stable carbon isotopes of bulk samples from different carbonate mounds from the same area, indicates that the sulphate-methane transition zone moves in depth through time. We will show and discuss multidisciplinary data obtained after several cruises aimed to elucidate the impact of microorganisms on the construction of these carbonate mounds. The special emphasis in this research will be on the correlation between microbial ecosystems and their metabolic influence on mineral formation and diagenesis

Biogeochemical evidence for anoxic oxidation of methane occurrences in the juvenile carbonate mounds from the Gulf of Cadiz

Carbonate mounds are conspicuous features of the European margins. Only fossil examples of these mounds were known when modern giant carbonate mounds were discovered in the S.-W. Irish margin. A decade of thorough studies in this area provided remarkable insight on mound processes and distribution. However, the question of the genesis and stabilization over geological times of these carbonate mounds remain wide open.Our work hypothesis is that moderate fluxes of low molecular hydrocarbons are oxidized and produces carbonates that may serve as cement for the mound stabilization. The recent discovery and mapping (R/V Belgica 2003, 2005) of the Pen Duick escarpment on the Moroccan margin (Gulf of Cadiz) by swath bathymetry shed light on new carbonate mounds associated with fluid migration markers such as pockmarks, carbonate crusts and mud volcanoes. Pore water biogeochemical profiles show that the sulphate to methane transition zone occurs at 3.5 meters below the sea floor within the mound, whereas the depth of no sulphate is much deeper in the surrounding sediments. At the same depth, carbonates are released with d13C values as low as -21 permil indicating a methane and possibly other light hydrocarbons origin. Hence anoxic oxidation of hydrocarbons, and subsequent carbonate production, may play a key role in the mound formation and/or stabilization. Interestingly, Lophelia coral rubbles were present all along the sediment column suggesting that this mound is a potential habitat for cold coral and associated communities

Integrated research on the Pen Duick cold-water coral mounds: the MiCROSYSTEMS approach

The ESF EuroDIVERSITY MiCROSYSTEMS project aimed to turn the cold-water coral (CWC) mounds on the Pen Duick Escarpment (PDE) in the Gulf of Cadiz into a natural laboratory, exploring this highly complex biotope and to characterize its biodiversity. A common point of discussion with all other CWC mound provinces, surpassing its broad range of regional and morphological variability, concerns the driving forces regarding the initiation of these complex deep-water systems. Both oceanographic and geological processes have been proposed to play a significant role in the mound nucleation, growth and decline. During IODP Expedition 307, the importance of biogeochemical processes was already elucidated. Here, we present the preliminary results of the MD169 campaign as an integrated case study of three PDE CWC mounds: Alpha, Beta and Gamma mounds.Although cold-water corals are a common feature on the adjacent cliffs, mud volcanoes and seafloor, no actual living reef has been observed during the many ROV surveys. This multidisciplinary study aims to present a comprehensive and holistic view on the local dynamic geological and oceanographic environment. Coring data suggests (past or present) methane seepage near the Pen Duick Escarpment. Several sources and pathways are proposed, among which a stratigraphic migration through uplifted Miocene series underneath PDE. Its dominant morphology has influenced the local hydrodynamics within the course of the Pliocene, as documented by the emplacement of a sediment drift. Predominantly during post-Middle Pleistocene glacial episodes, favourable conditions were present for mound growth. An additional advantage for CWC mound nucleation near the top of PDE is offered through seepage-related carbonate crusts which might offer elevated colonization positions. Present-day seabed observations also suggested a possible important role of open coral rubble frameworks in the mound building process. These graveyards not only act as sediment trap but also as micro-habitat for a wide range of organisms. The presence of a fluctuating Sulphate-Methane Transition Zone is responsible for diagenesis, affecting both geochemical as physical characteristics, transforming the buried reef into a solid mound. Nevertheless, these seepage fluxes seem to be locally variable. As such, the origin and evolution of the PDE CWC mounds is, probably more than any other NE Atlantic cold-water coral mound province, located on the crossroads of environmental (hydrodynamic) and geological (seepage) pathways