Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Related Publications
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Geochemical Analyses:
Sulfate concentration measurements were completed shipboard; after centrifuging sediment-filled 15-milliliter (ml) tubes, the overlying porewater was filtered through 0.45-micrometer (um) filters, acidified with 50 microliters (ul) of 50% HCl and bubbled with nitrogen for 4 minutes to remove sulfide. Sulfate concentrations were then measured shipboard using a 2010i Dionex Ion Chromatograph (Sunnyvale, CA, USA) through Ag exchange columns (Dionex) so as to remove Cl (Martens et al., 1999). For sulfide, 1 ml porewater samples were combined with 0.1 molar (M) zinc acetate and concentrations were analyzed spectrophotometrically on the ship (Cline, 1969).

Headspace methane concentrations were determined onboard by standard gas chromatography with a flame ionization detector (FID), specifically using a HACH Carle Series 100 AGC Gas Chromatograph (GC) with an Alltech Molecular Sieve 5A packed column (80/100 mesh, 3.05 meters (m) length, 3.2 millimeter internal diameter (mm ID)) and an 80 degree Celsius (C) isothermal temperature profile. Stable isotopic compositions of the same methane samples were measured post-cruise at the University of North Carolina (UNC) via gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) on a Finnigan MAT 252 Isotope Ratio Mass Spectrometer, using an HP 5890 Series II Gas Chromatograph with an HP Plot Q column (30 m length, 0.32 mm ID, 20 um film thickness) and a 30 degree C isothermal temperature profile.

To measure DIC, 2 ml of unamended porewater from each sediment horizon were injected into evacuated serum vials (30 ml) and stored upside down at -20 degrees C. At UNC, the samples were thawed, and DIC was reacted to gaseous CO2 by adding 1 ml of a 30% phosphoric acid solution to each serum vial and shaking vigorously before GC analysis (Kelley et al., 1990). Stable isotopic values and concentrations of DIC were analyzed via coupled GC (Hewlett Packard 5890) and Isotope Ratio Mass Spectrometer (Finnigan MAT 252).

Porewater concentrations of dissolved organic acids were measured via High-Pressure Liquid Chromatography (HPLC) after the cruise, using a Beckman Model 332 gradient liquid chromatograph combined with an ISCO V4 UV/VIS detector and a Shimadzu CR3-A integrator (Albert and Martens, 1997).

BCO-DMO Processing Notes:
- combined the following original files/sheets into one dataset:
-- sheet named "Annotated clean data" of file named "guaymas methane 2009.xls"
-- sheet named "Clean Data" of file named "Guaymas DIC 2009.xls"
-- sulfate and sulfide data in file named "Sulfate_sulfide_temperatures 2009.xlsx" (re-organized from separate sheets into one table)
- renamed fields to comply with BCO-DMO naming conventions.

While microbial communities in marine sediments are generally sustained by sedimentation of organic matter from the water column, the Guaymas Basin hydrothermal sediments provide a model system for the microbial utilization and transformation of thermally released microbial substrates from deeply buried marine organic matter. Thermal generation of subsurface organic carbon compounds is usually restricted to deeply buried subsurface sediments, where it sustains deep subsurface microbiota. However, in the Guaymas Basin, the thermally generated organic substrates of subsurface origin fuel a complex microbial ecosystem in surficial sediments that can be sampled by submersible. As a working hypothesis, the physiologically distinct, layered microbial communities force the geothermally produced substrates through a double “microbial gauntlet” of anaerobic metabolism and autotrophic carbon fixation, where terminal anaerobic degradation of organic matter is performed by methanogenic and methane-oxidizing archaea, by sulfate-reducing bacteria and archaea, and (to be tested) by novel subsurface archaeal populations within the upper sediments, while inorganic and organic remineralization products are assimilated by sulfur-oxidizing Beggiatoa mats at the sediment surface. We aim at a quantitative understanding of how the dense and highly active benthic microbial populations of the Guaymas system utilize and recycle organic and inorganic carbon and sulfur of subsurface origin, how geochemical controls affect the community structure, and how uncultured, globally occurring subsurface archaea and bacteria thrive in their sediment habitat. More generally, microbial utilization and recycling of deeply buried, fossil carbon and sulfur in benthic sediments and the sedimentary subsurface is a “seldom seen“ but essential part of these microbially driven processes in the marine biosphere. To analyze the complex interplay of thermogenic and biogenic carbon sources and sinks, and the role of uncultured microbial populations in these processes, geochemical and molecular-biological approaches are integrated and combined. The microbial community composition and activity patterns will be analyzed quantitatively (rRNA membrane slot blot hybridization; single-strand rRNA conformation polymorphism) and with qualitative diversity surveys (PCR, cloning and sequencing). Carbon assimilation patterns in specific functional and phylogenetic groups of prokaryotes will be analyzed using carbon-isotopic analysis of ribosomal RNA, intact polar lipids, and whole microbial cells (using FISH-SIMS). Carbon substrate profiles and microbial process rates (sulfate reduction, methanogenesis, methane oxidation) across hydrothermally active sediment sites and down-core will correlate microbial populations and substrate utilization. Stable carbon isotopic analysis of key microbial substrates will further constrain the microbial utilization patterns of isotopically distinct carbon pools in specific sediment layers.

To summarize, in situ and lab results indicate that newly discovered, phylogenetically distinct populations of Anaerobic Methane-oxidizing archaea (ANMEs) in Guaymas Basin, and their presumed syntrophic bacterial partners, are capable of methane oxidation at high temperatures, at least up to 70-75°C. Isotopically light carbon (indicative of a methane-derived contribution) permeates into sedimentary microbial populations and microbial mats in hydrothermally active areas, as shown by 13C analysis of extracted bacterial and archaeal rRNA. Manipulative incubations with Guaymas sediments suggest a mode of anaerobic methane oxidation which appears to operate uncoupled to sulfate reduction, and requires near in situ methane concentration. Rigorous testing is required for validation of the process and identification of the organisms responsible. High-temperature tolerant and sulfate-uncoupled anaerobic methane oxidation require re-evaluation of the classical controls of this process, temperature and sulfate availability.

By installing autonomous temperature loggers in Guaymas sediments covered with Beggiatoa spp. mats, we have obtained continuous temperature profiles, from the sediment surface to 40 cm depth, over up to 11 days. In contrast to previous one-time temperature measurements that provided only a static snapshot, these data revealed substantial temperature fluctuations in the upper cm layers underlying orange Beggiatoa mats, indicative of fluctuations in hydrothermal flux and/or advective in-mixing of seawater. Such temperature regimes would select for eurythermal bacteria and archaea that tolerate a broad mesophilic/thermophilic temperature range, or for microbial communities that consist of members with different temperature optima, that co-occur or overlap in the same sediment layer but vary in activity depending on temperature and associated geochemical conditions.

Anaerobic microbial processes in sediments (sulfate reduction, remineralization of biomass, anaerobic methane oxidation) produce DIC and sulfide that, in turn, sustain the Beggiatoa mats, assuming autotrophic capability. To examine this link between sediment processes and surface mats, we quantified temperature gradients, porewater concentration gradients (sulfide, sulfate, methane, DIC, volatile organic acids), and 13C-isotopic signatures of methane and DIC underneath orange and white Beggiatoa mats (differentiated by 16S rRNA sequencing), and the bare sediment. The steepest temperature and porewater concentration gradients (sulfide and DIC) are mostly found under orange Beggiatoa mats that occur in the center of Beggiatoa patches. Temperature and geochemical gradients are attenuated under white Beggiatoa mats, which surround the orange mats in a sunny-side up pattern, and flatten out or disappear in the surrounding mat-free sediment

We are annotating the genome of an orange Beggiatoa spp. from Guaymas Basin [taxonomically revised as Maribeggiatoa], recovered from a single filament after whole genome amplification. Sequencing was completed at JCVI, supported by the Gordon and Betty Moore Foundation. The single-filament genome is not completely assembled, but is of approximately the expected total length and includes a full complement of ribosomal protein, tRNA, and tRNA synthetase genes. So far, the genome content is broadly consistent with a nitrate-reducing, facultatively autotrophic sulfur-oxidizing bacterium.

Publications associated with this project are as follows:
Note: this is now a list of all publications that use samples collected from the NSF-funded Guaymas cruises AT15-40 and AT15-56. All these publications were funded from NSF award OCE-0647633, the grant that funded these two cruises. Those publications that were written and published after 2013 continue to use samples collected and analyzed on cruises AT15-40 and AT15-56 under NSF award OCE-0647633, but the effort in analyzing the data and writing the manuscript also relied on funding by OCE-1357238. Since we will not have new samples until late in 2016, current work and publications on OCE-1357238 will continue to rely on samples collected during cruises AT15-40 and AT15-56.

Holler, T. F. Widdel, K. Knittel, R. Amann, M. Y. Kellermann, K.-. Hinrichs, A. Teske, A. Boetius, and G. Wegener. 2011. Thermophilic anaerobic oxidation of methane by marine microbial consortia. The ISME Journal 5:1946-1956. doi:10.1038/ismej.2011.77

Biddle, J.F., Z. Cardman, H. Mendlovitz, D.B. Albert, K.G. Lloyd, A. Boetius, and A. Teske. 2012. Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments. The ISME Journal 6:1018-1031. doi:10.1038/ismej.2011.164

McKay, L.J., B.J. MacGregor, J.F. Biddle, H.P. Mendlovitz, D. Hoer, J.S. Lipp, K.G. Lloyd, and A.P. Teske. 2012. Spatial heterogeneity and underlying geochemistry of phylogenetically diverse orange and white Beggiatoa mats in Guaymas Basin hydrothermal sediments. Deep-Sea Research I, 67:21-31. doi:10.1016/j.dsr.2012.04.011

Bowles, M.W., L.M. Nigro, A.P. Teske, and S.B. Joye.. 2012. Denitrification and environmental factors influencing nitrate removal in Guaymas Basin hydrothermally-altered sediments. Frontiers in Microbiology 3:377. doi:10.3389/fmicb.2012.03377

MacGregor, B.J., J.F. Biddle, J.R. Siebert, E. Staunton, E. Hegg, A.G. Matthysse, and A. Teske. 2013. Why orange Guaymas Basin Beggiatoa spp. are orange: Single-filament genome-enabled identification of an abundant octaheme cytochrome with hydroxylamine oxidase, hydrazine oxidase and nitrite reductase activities. Applied and Environmental Microbiology 79:1183-1190. doi:10.1128/AEM.02538-12

MacGregor, B.J., J.F. Biddle, and A. Teske. 2013. Mobile elements in a single-filament orange Guaymas Basin Beggiatoa ("Candidatus Maribeggiatoa") sp. draft genome; evidence for genetic exchange with cyanobacteria. Applied and Environmental Microbiology 79:3974-3985. doi:10.1128/AEM.03821-12

Meyer, S., G. Wegener, K.G. Lloyd, A. Teske, A. Boetius, and A. Ramette. 2013. Microbial habitat connectivity across spatial scales and hydrothermal temperature gradients at Guaymas Basin. Frontiers in Microbiology 4:207. doi:10.3389/fmic.2013.00207

MacGregor, B.J., J.F. Biddle, C. Harbort, A.G. Matthysse, and A. Teske. 2013. Sulfide oxidation, nitrate respiration, carbon acquisition and electron transport pathways suggested by the draft genome of a single orange Guaymas Basin Beggiatoa (Cand. Maribeggiatoa) sp. filament. Marine Genomics 11:53-65. doi:10.1016/j.margen.2013.08.001

Ruff, E., J.F. Biddle, A. Teske, K. Knittel, A. Boetius, and A. Ramette. 2015. Global dispersion and local diversification of the methane seep microbiome. Proc. Natl. Acad. Sci. USA, 112:4015-4020. doi:10.1073/pnas.1421865112

McKay, L., V. Klokman, H. Mendlovitz, D. LaRowe, M. Zabel, D. Hoer, D. Albert, D. de Beer, J. Amend, A. Teske. Thermal and geochemical influences on microbial biogeography in the hydrothermal sediments of Guaymas Basin. Environmental Microbiology, in revision.

Dowell, F., Z. Cardman, S. Dasarathy, M.Y. Kellermann, L.J. McKay, B.J. MacGregor, S.E. Ruff, J.F. Biddle, K.G. Lloyd, J.S. Lipp, K-U. Hinrichs, D.B. Albert, H. Mendlovitz, and A. Teske. Microbial communities in methane and short alkane-rich hydrothermal sediments of Guaymas Basin. Frontiers in Microbiology, In Revision.

Conference abstracts (post 2013, only NSF-OCE 1357238):
B.J. MacGregor. 2014. Receiver (REC) domains in the orange Guaymas "Maribeggiatoa" (BOGUAY) draft genome: an evolutionary network of sensor networks. The Human and Environmental Microbiome Symposium 2014. Duke Center for the Genomics of Microbial Systems, Durham, NC.

B.J. MacGregot. 2015. Abundant intergenic repeats and a possible alternate RNA polymerase betra subunit in the orange Guaymas "Maribeggiatoa" genome. American Society for Microbiology 2015 General Meeting. New Orleans, LA.

Z. Cardman, L.J. McKay, E. Dowell, S. Dasarathy, V. Klokman, J.F. Biddle, K.G. Lloyd, H. Mendlovitz, D. Albert, M. Kellermann, K.-U. Hinrichs, B.J. MacGregir and A.P. Teske. 2014. American Society for Microbiology 2014 General Meeting. Boston, MA.