Person: Victoria J. Orphan

Abstract

The past decade of scientific ocean drilling has revealed seemingly ubiquitous, slow-growing microbial life within a range of deep biosphere habitats. Integrated Ocean Drilling Program Expedition 337 expanded these studies by successfully coring Miocene-aged coal beds 2 km below the seafloor hypothesized to be “hot spots” for microbial life. To characterize the activity of coal-associated microorganisms from this site, a series of stable isotope probing (SIP) experiments were conducted using intact pieces of coal and overlying shale incubated at in situ temperatures (45 °C). The 30-month SIP incubations were amended with deuterated water as a passive tracer for growth and different combinations of ¹³C- or ¹⁵N-labeled methanol, methylamine, and ammonium added at low (micromolar) concentrations to investigate methylotrophy in the deep subseafloor biosphere. Although the cell densities were low (50–2,000 cells per cubic centimeter), bulk geochemical measurements and single-cell–targeted nanometer-scale secondary ion mass spectrometry demonstrated active metabolism of methylated substrates by the thermally adapted microbial assemblage, with differing substrate utilization profiles between coal and shale incubations. The conversion of labeled methylamine and methanol was predominantly through heterotrophic processes, with only minor stimulation of methanogenesis. These findings were consistent with in situ and incubation 16S rRNA gene surveys. Microbial growth estimates in the incubations ranged from several months to over 100 y, representing some of the slowest direct measurements of environmental microbial biosynthesis rates. Collectively, these data highlight a small, but viable, deep coal bed biosphere characterized by extremely slow-growing heterotrophs that can utilize a diverse range of carbon and nitrogen substrates.

Abstract

The anaerobic oxidation of methane by anaerobic methanotrophic (ANME) archaea in syntrophic partnership with deltaproteobacterial sulfate-reducing bacteria (SRB) is the primary mechanism for methane removal in ocean sediments. The mechanism of their syntrophy has been the subject of much research as traditional intermediate compounds, such as hydrogen and formate, failed to decouple the partners. Recent findings have indicated the potential for extracellular electron transfer from ANME archaea to SRB, though it is unclear how extracellular electrons are integrated into the metabolism of the SRB partner. We used metagenomics to reconstruct eight genomes from the globally distributed SEEP-SRB1 clade of ANME partner bacteria to determine what genomic features are required for syntrophy. The SEEP-SRB1 genomes contain large multiheme cytochromes that were not found in previously described free-living SRB and also lack periplasmic hydrogenases that may prevent an independent lifestyle without an extracellular source of electrons from ANME archaea. Metaproteomics revealed the expression of these cytochromes at in situ methane seep sediments from three sites along the Pacific coast of the United States. Phylogenetic analysis showed that these cytochromes appear to have been horizontally transferred from metal-respiring members of the Deltaproteobacteria such as Geobacter and may allow these syntrophic SRB to accept extracellular electrons in place of other chemical/organic electron donors.

Abstract

Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range of Deltaproteobacteria diversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seep sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. Many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1, Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involving Epsilon-, Delta-, and Gammaproteobacteria in methane seep ecosystems.

Abstract

This study examines the potential for the biologically mediated anaerobic oxidation of methane (AOM) coupled to sulfate reduction on ancient Mars. Seven distinct fluids representative of putative martian groundwater were used to calculate Gibbs energy values in the presence of dissolved methane under a range of atmospheric CO₂ partial pressures. In all scenarios, AOM is exergonic, ranging from −31 to −135 kJ/mol CH₄. A reaction transport model was constructed to examine how environmentally relevant parameters such as advection velocity, reactant concentrations, and biomass production rate affect the spatial and temporal dependences of AOM reaction rates. Two geologically supported models for ancient martian AOM are presented: a sulfate-rich groundwater with methane produced from serpentinization by-products, and acid-sulfate fluids with methane from basalt alteration. The simulations presented in this study indicate that AOM could have been a feasible metabolism on ancient Mars, and fossil or isotopic evidence of this metabolic pathway may persist beneath the surface and in surface exposures of eroded ancient terrains.

Abstract

IODP Expedition 337 set the record for deepest marine scientific drilling down to 2.4 kmbsf. This cruise also had the unique opportunity to retrieve deep cores from the Shimokita coal bed system in Japan with the aseptic and anaerobic conditions necessary to look for deep life. Onboard scientists prepared nearly 1,700 microbiology samples shared among five different countries to study life in the deep biosphere. Samples spanned over 1km in sampling depths and include representatives of shale, sandstone, and coal lithologies. Findings from previous IODP and deep mine expeditions suggest the genetic potential for methylotrophy in the deep subsurface, but it has yet to be observed in incubations. A subset of Expedition 337 anoxic incubations were prepared with a range of ¹³C-methyl substrates (methane, methylamine, and methanol) and maintained near in situ temperatures. To observe ¹³C methyl compound metabolism over time, we monitored the δ¹³C of the dissolved inorganic carbon and methane (by-products of methyl compound metabolism) over a period of 1.5 years. Our geochemical evidence suggests that the coal horizon incubated with ¹³C-methylamine showed the highest activity of all methyl incubations. Therefore, there are not only cells in the deeply buried terrigenous coal bed at Shimokita, but a microbial community that can be activated by methylotrophic compounds. Incubations showing the highest geochemical activity were prepared at the JAMSTEC Kochi Core Center for nanoSIMS analysis in March of 2015, and will be analyzed at Caltech in the coming months. This will allow us to observe if cells also incorporated the labeled methyl compounds into their body mass and provide another line of evidence that these substrates were used by the deep coalbed microbial community.

Abstract

The very low rates of activity typically observed for subseafloor microbes make it hard to differentiate slow-growing from inactive cells. During his C-DEBI postdoctoral fellowship Roland Hatzenpichler developed novel approaches to detect biosynthetic activity in uncultured cells directly in their habitat. Based on the incorporation of synthetic amino acids into new proteins and their subsequent detection via fluorescence staining (BONCAT, bioorthogonal non-canonical amino acid tagging), protein synthesis active cells are visualized via epifluorescence microscopy. In combination with fluorescence-activated cell-sorting, they can then be separated from complex samples. After adapting BONCAT for environmental applications, Roland used BONCAT to track the in situ translational activity of syntrophic archaeal-bacterial consortia catalyzing the anaerobic oxidation of methane in deep-sea sediments (Hatzenpichler et al., PNAS 2016). By combining sorting of active consortia, whole genome amplification, and 16S rRNA gene sequencing, the identities of hundreds of individual, biosynthetically active cellular partnerships could be resolved. This approach revealed that representatives of all major methane-oxidizing archaeal clades were active under controlled incubation conditions. Unexpectedly, some consortia were anabolically active even in the absence of methane, suggesting energy sources beyond methane. It also led to the discovery of a previously unrecognized interaction of methane-oxidizing archaea with members of the environmentally highly abundant, yet poorly understood phylum Verrucomicrobia. Since November 1^st 2016, Roland is an Assistant Professor in the Department of Chemistry and Biochemistry at Montana State University, Bozeman. His group focuses on the ecophysiology and in situ activity of uncultured archaea in a variety of sediment environments. www.environmental-microbiology.com

Abstract

Although the subsurface biosphere is now recognized as an important reservoir of life on our planet, until recently the microbial community beneath open-ocean oligotrophic gyres (making up the majority of the seafloor) has not been studied in detail (D’Hondt et al., 2004, 2009). IODP Expedition 329 has taken a first step at characterizing the microbial community beneath the South Pacific Gyre. This region has low biological surface productivity and therefore very low organic carbon burial rates (10^-8 and 10^-10 moles C cm^-1 yr^-1), deep oxygen penetration (sediments are oxidized to the basement), and low prokaryotic cell counts (10⁶ cells cm^-3 to <10³ cells cm^-3) (D’Hondt et al., 2009; Fischer et al., 2009, IODP Exp. 329 Preliminary Report, 2011). In these sediments, the dominant fraction of organic carbon may be aggregated or adsorbed to minerals (Arnarson & Kiel 2007). Thus the ability to colonize minerals should be an important ecological adaptation, with those microbes that are able to grow on the minerals creating potential “hotspots” of microbial activity within these oligotrophic sediments. Our project aims to determine whether there is stimulated microbial activity associated in long-term incubations with H¹³CO^3- and ¹⁵NO^3-. Specific mineral and clay fractions in the oligotrophic South Pacific Gyre sediment system were targeted using combination of magnetic and density separation and SEM-EDS. The bacterial and archaeal community were examined by CARD-FISH, CARD-FISH-nanoSIMS, and 16S rRNA tag sequencing. Overall results from this C-DEBI grant have shown the viability of magnetic separation and identification of single cells in subsurface sediments as a method for investigating mineral association in microbial communities. We have identified putatively viable cells attached to 7 Fe/Mn-rich minerals, potentially representing an unexplored strategy for low-carbon environments. We also have discovered a higher level of diversity in the paramagnetic (Fe/Mn-rich) mineral-associated bacteria and higher number of Marine Group I archaeal OTUs compared to the diamagnetic fraction in the oligotrophic subsurface sediment from the South Pacific Gyre.