Evidence for microbial iron reduction in the methanic sediments of the oligotrophic southeastern Mediterranean continental shelf

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Vigderovich, H., Liang, L., Herut, B., Wang, F., Wurgaft, E., Rubin-Blum, M., & Sivan, O. Evidence for microbial iron reduction in the methanic sediments of the oligotrophic southeastern Mediterranean continental shelf. Biogeosciences, 16(16), (2019): 3165-3181, doi: 10.5194/bg-16-3165-2019.Dissimilatory iron reduction is probably one of the oldest types of metabolisms that still participates in important biogeochemical cycles, such as those of carbon and sulfur. It is one of the more energetically favorable anaerobic microbial respiration processes and is usually coupled to the oxidation of organic matter. Traditionally this process is thought to be limited to the shallow part of the sedimentary column in most aquatic systems. However, iron reduction has also been observed in the methanic zone of many marine and freshwater sediments, well below its expected zone and occasionally accompanied by decreases in methane, suggesting a link between the iron and the methane cycles. Nevertheless, the mechanistic nature of this link (competition, redox or other) has yet to be established and has not been studied in oligotrophic shallow marine sediments. In this study we present combined geochemical and molecular evidences for microbial iron reduction in the methanic zone of the oligotrophic southeastern (SE) Mediterranean continental shelf. Geochemical porewater profiles indicate iron reduction in two zones, the uppermost part of the sediment, and the deeper zone, in the layer of high methane concentration. Results from a slurry incubation experiment indicate that the deep methanic iron reduction is microbially mediated. The sedimentary profiles of microbial abundance and quantitative PCR (qPCR) of the mcrA gene, together with Spearman correlation between the microbial data and Fe(II) concentrations in the porewater, suggest types of potential microorganisms that may be involved in the iron reduction via several potential pathways: H2 or organic matter oxidation, an active sulfur cycle, or iron-driven anaerobic oxidation of methane. We suggest that significant upward migration of methane in the sedimentary column and its oxidation by sulfate may fuel the microbial activity in the sulfate methane transition zone (SMTZ). The biomass created by this microbial activity can be used by the iron reducers below, in the methanic zone of the sediments of the SE Mediterranean.This study was supported by the joint grant of Israel Science Foundation and the National Natural Science Foundation of China (ISF-NSFC) (grant numbers 31661143022 (FW) and 2561/16 (OS)). Funding was provided to Hanni Vigderovich by the Mediterranean Sea Research Center of Israel

Hydrocarbon-related microbial processes in the deep sediments of the Eastern Mediterranean Levantine Basin

During the 2011 exploration season of the EV Nautilus in the Mediterranean Sea, we conducted a multidisciplinary study, aimed at exploring the microbial populations below the sediment–water interface (SWI) in the hydrocarbon-rich environments of the Levantine basin. Two c. 1000-m-deep locations were sampled: sediments fueled by methane seepage at the toe of the Palmachim disturbance and a patch of euxinic sediment with high sulfide and methane content offshore Acre, enriched by hydrocarbon from an unknown source. We describe the composition of the microbial population in the top 5 cm of the sediment with 1 cm resolution, accompanied by measurements of methane and sulfate concentrations, and the isotopic composition of this methane and sulfate (δ13CCH4, δ18OSO4, and δ34SSO4). Our geochemical and microbiological results indicate the presence of the anaerobic methane oxidation (AOM) coupled to bacterial sulfate reduction (BSR). We show that complex methane and sulfur metabolizing microbial populations are present in both locations, although their community structure and metabolic preferences differ due to potential variation in the hydrocarbon source

Fueled by methane: deep-sea sponges from asphalt seeps gain their nutrition from methane-oxidizing symbionts

Sponges host a remarkable diversity of microbial symbionts, however, the benefit their microbes provide is rarely understood. Here, we describe two new sponge species from deep-sea asphalt seeps and show that they live in a nutritional symbiosis with methane-oxidizing (MOX) bacteria. Metagenomics and imaging analyses revealed unusually high amounts of MOX symbionts in hosts from a group previously assumed to have low microbial abundances. These symbionts belonged to the Marine Methylotrophic Group 2 clade. They are host-specific and likely vertically transmitted, based on their presence in sponge embryos and streamlined genomes, which lacked genes typical of related free-living MOX. Moreover, genes known to play a role in host–symbiont interactions, such as those that encode eukaryote-like proteins, were abundant and expressed. Methane assimilation by the symbionts was one of the most highly expressed metabolic pathways in the sponges. Molecular and stable carbon isotope patterns of lipids confirmed that methane-derived carbon was incorporated into the hosts. Our results revealed that two species of sponges, although distantly related, independently established highly specific, nutritional symbioses with two closely related methanotrophs. This convergence in symbiont acquisition underscores the strong selective advantage for these sponges in harboring MOX bacteria in the food-limited deep sea

Massive asphalt deposits, oil seepage, and gas venting support abundant chemosynthetic communities at the Campeche Knolls, southern Gulf of Mexico

Hydrocarbon seepage is a widespread process at the continental margins of the Gulf of Mexico. We used a multidisciplinary approach, including multibeam mapping and visual seafloor observations with different underwater vehicles to study the extent and character of complex hydrocarbon seepage in the Bay of Campeche, southern Gulf of Mexico. Our observations showed that seafloor asphalt deposits previously only known from the Chapopote Knoll also occur at numerous other knolls and ridges in water depths from 1230 to 3150 m. In particular the deeper sites (Chapopopte and Mictlan knolls) were characterized by asphalt deposits accompanied by extrusion of liquid oil in form of whips or sheets, and in some places (Tsanyao Yang, Mictlan, and Chapopote knolls) by gas emission and the presence of gas hydrates in addition. Molecular and stable carbon isotopic compositions of gaseous hydrocarbons suggest their primarily thermogenic origin. Relatively fresh asphalt structures were settled by chemosynthetic communities including bacterial mats and vestimentiferan tube worms, whereas older flows appeared largely inert and devoid of corals and anemones at the deep sites. The gas hydrates at Tsanyao Yang and Mictlan Knolls were covered by a 5-to-10 cm-thick reaction zone composed of authigenic carbonates, detritus, and microbial mats, and were densely colonized by 1–2 m-long tube worms, bivalves, snails, and shrimps. This study increased knowledge on the occurrences and dimensions of asphalt fields and associated gas hydrates at the Campeche Knolls. The extent of all discovered seepage structure areas indicates that emission of complex hydrocarbons is a widespread, thus important feature of the southern Gulf of Mexico

CLSM reconstruction of bacteria in embryo tissue of deep-sea sponge H. (S.) methanophila, EUB probe

Catalyzed reporter deposition (CARD)-FISH with horseradish peroxidase-labeled probes (Biomers, Ulm, Germany), EUB338 (Amann et al.,1990) was performed on 8 μm sections of sponge tissue. Photomicrographs were acquired with confocal laser-scanning microscope (LSM 780, Carl Zeiss, Germany). Three channels were used - cy3, EUB probe, DAPI and 488 (autofluoresence). 3D representation of image stacks was reconstructed with Fiji 3D project option (interpolated).

Additional FISH images of methanotrophic (MOX, MITC849 probe double-labelled with 594 dye) symbionts in 10 μm sections of H. (S.) methanophila sponge (Mictlan Knoll individual), epifluorescence microscpopy

We double-labelled MTC849 (5` – CGTTAGCTCCACCACTAAG – 3`) with Atto594 dye (Biomers, Ulm, Germany), and hybridized it with sponge tissue in 20% formamide buffer using standard protocol (Duperron <i>et al.</i>, 2008). Photomicrographs were acquired with Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Jena, Germany), 40x or 100x lenses (see file name). Three channels were used - 594 (MITC849), DAPI and FITC(autofluoresence)

Fluorescence in situ hybridization (FISH) images of methanotrophic (MOX, MITC849 probe double-labelled with 594 dye) symbionts in 10 μm sections of H. (S.) methanophila sponge (Mictlan Knoll individual).

Twenty 3-channel images were acquired with Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Jena, Germany) with 10x magnification. Color legend is: MOX, magenta; DNA (DAPI), blue; autofluoresence at ~520 nm with ~490 nm excitation (FITC filter), green. These images were aligned with FIJI Grid/collection stitching plugin to produce the resulting mosaic. <br

Fluorescence in situ hybridization (FISH) images of methanotrophic (MOX, MITC849 probe double-labelled with 594 dye) symbionts in 10 μm sections of I. methanophila sponge (Chapopote Knoll).

We double-labelled MTC849 (5` – CGTTAGCTCCACCACTAAG – 3) with Atto594 dye (Biomers, Ulm, Germany), and hybridized it with sponge tissue in 20% formamide buffer using standard protocol (Duperron <i>et al.</i>, 2008). Photomicrographs were acquired with confocal laser-scanning microscope (LSM 780, Carl Zeiss, Germany) - named CLSM*** or with Zeiss Axioplan 2 epifluorescence microscope (Zeiss, Jena, Germany) - named 7-108-ROV17*** . Three channels were acquired - 594, DAPI and 488/FITC (autofluoresence). Five images that had been acquired with 10x magnification have been used to reconstruct the overview representation