URLhttps://www.bco-dmo.org/dataset/712652
Download URLhttps://www.bco-dmo.org/dataset/712652/data/download
Media Typetext/tab-separated-values
CreatedAugust 10, 2017
ModifiedAugust 2, 2019
StateFinal no updates expected
Brief DescriptionIODP Expedition 337 single cell isotope incorporation of 1H, 2H, 12C14N, 12C15N. 12C12C, 12C13C ions

Acquisition Description

SIP Incubation Preparation
IODP Expedition 337 operations commenced July 26 and continued through September 30, 2012 on the D/V Chikyu. Utilizing riser drilling, a sedimentary sequence was recovered down to 2466 m below seafloor (mbsf) at Hole C0020A (41 10' 36" N, 142 12' 02" E) in 1180 m water depth off the Shimokita Peninsula. The drilled sequence transitioned from open marine (youngest; late Pliocene, ~5 Ma) to terrestrial (oldest; late Oligocene, ~30 Ma) with depth. Models for maximum temperature reached by Expedition 337 coring report 63.7 degrees C. Shipboard sedimentological, geochemical, and microbiological data and methods are available through IODP publications. Additional coal petrography is available in Gross et al.  A total of 52 incubation amendment conditions were prepared onboard to interrogate a range of potential deep-biosphere metabolic strategies, and then incubated back in the lab at temperatures approximating that measured in situ. In this study, incubations from shale (Core 8L4; 1606 mbsf; 37C incubation temperature), coal (Core 15R3; 1921 mbsf; 45C incubation temperature), and mixed (homogenized mixture from multiple cores 19R1, 19R5, 19R7, 20R3, 23R6, 23R8, 24R3, 25R1, 25R2, and 25R3; 1950-1999 mbsf; 45C incubation temperature) with methanol and methylamine substrate additions were analyzed. Age estimates of these samples are early to middle Miocene. In situ temperatures ranged from 38C to 48C at these sample depths with pressures ~30 MPa. Two coal beds were included in these incubations: a shallower coal-only sample deposited under more marine-influenced conditions (~1921 mbsf, core 15R3) and a deeper coal bed deposited under more limnic conditions that was included in the mixed lithology sample (~2000 mbsf, cores 24R3 and 25R1). Cores used for incubations were prepared by removal of outer drill-fluid-contaminated layers by sterile ceramic knife as soon as possible after core recovery and stored at 4C until incubation preparation, while maintaining an anaerobic atmosphere during the entire process. For preparation of the SIP incubations, the interior portion of the core was manually crushed into cm-sized pieces under sterile, anaerobic conditions and distributed evenly into sterile 50 ml glass vials with butyl rubber stoppers and screw caps (Nichidenrika-Glass Co. Ltd.).  Vials were flushed with argon and pressurized to 1 atm argon headspace. Sterile C-, N-, and S-free media (1% PBS, 30 g/L NaCl, 12 g/L MgCl2, and 3 g/L KCl) was prepared anaerobically with deuterated water (20 at. % 2H2O). 20 at. % 2H2O was selected as the highest level of enrichment with little to no effect on the activity of microorganisms in pure culture. Time point 1, time point 2, and autoclaved treatments were prepared for each substrate condition. Time point 1 incubations lasted for six months, while time point 2 and autoclaved treatments were maintained at the in situ incubation temperature for 2.5 years. Due to low levels of activity ascertained from geochemical measurements, all NanoSIMS analyses were conducted on time point 2 and autoclaved samples. Amendments and incubation conditions for the methyl-substrate subset analyzed in this study are provided in this dataset. Equimolar amounts of substrate (30 umol C, 1.5 mM final; 3 umol N, 0.15 mM final) were added across incubation conditions at 50 at. % (Cambridge Isotopes). Hydrogen was added as 5 mL 100% H2 overpressure to incubations (~15% H2 headspace). A full list of the additional incubation conditions prepared onboard are listed in cruise Methods. Alkalinity (34.39 – 9.68 mM) ammonium (2.80 – 1.83 mM) concentrations from formation fluid samples collected onboard exceed concentrations of C and N amendments. Concentrations of methylamine (0.05 mM) and methanol (1 mM) measured from lignite coal also suggest our substrate additions were environmentally relevant. After 30 months of incubation (March 2014) all treatments were sampled for geochemical analyses prior to preparation for NanoSIMS. 3 ml of headspace gas was removed to a vial filled with 0.1 M NaOH for methane analysis. About 1 ml of liquid was filtered through a 0.1 um 13 mm Whatman Polycarbonate Nuclepore Track-Etched Membrane (110405) for DIC analysis. See Supplemental Methods of Trembath-Reichert et al. for detailed description of methane and DIC analyses.

Sample preparation for NanoSIMS analysis
To overcome technical challenges for NanoSIMS analysis of low biomass samples, cell separation and fluorescence-activated cell sorting (FACS) were used to directly concentrate cells in a small analysis area, ~1 to 0.5 sq. mm. NanoSIMS samples were prepared from paraformaldehyde (PFA)-fixed cell separates after 894 days of incubation. Cell preservation, separation, enumeration, and FACS were all conducted in the clean booth and clean room facilities at Kochi Institute for Core Sample Research, JAMSTEC. Half of the solid and half of the liquid portion of each sample were fixed overnight in a solution of 2% paraformaldehyde (PFA), 3 × phosphate buffered saline (PBS). Samples were then subjected to two washes, incubating in 3 × PBS for 6 hrs and then 2 hrs, after each wash respectively. Samples were centrifuged (3500 × g) and supernatant was decanted after each wash. PFA-fixed samples were stored in 50 % ethanol : 3 × PBS. The other half of the sample was preserved in glyTE (70% glycerol, 100mM Tris, 10mM EDTA; Bigelow Single Cell Genomics Center preservation protocol) and frozen by cell alive system (CAS) and stored at -80C. 1 ml liquid and ~1 g sediment chips were subsampled by pipet and sterile cell culture loop, respectively, from the PFA-fixed sample. Cell separation, microscopy, and sorting procedures followed Morono et al., with the following modifications: 1) samples were sonicated (Bioruptor UCD-250, COSMO BIO) in an ice bath for 20 cycles of 30 sec 200 W, 30 sec off, and 2) samples were incubated in hydrofluoric acid post initial sonication, rather than after first density gradient separation. Cell detection limit was determined by no-sample added controls run in parallel with samples. Cells were stained with SYBR Green I (1:40 dilution of SYBR Green in Tris (10 mM) –EDTA (1mM) (TE) and sorted following the flow cytometry protocol of Morono et al. Sorted cells were concentrated directly from the sorter onto NanoSIMS compatible 0.2 um polycarbonate filters coated with indium tin oxide (ITO) as described in Morono et al. and Inagaki et al. ITO coating on polycarbonate membranes (Isopore GTBP02500 Millipore) was prepared by sputtering deposition technique at Astellatech Co. Ltd. (Kanagawa, Japan). Scanning electron microscopy (SEM) of the filters was done on a Zeiss 1550 VP Field Emission Scanning Electron Microscope at the GPS Division Analytical Facility at Caltech and SYBR stained cells were imaged with a BX51 epifluorescence microscope (Olympus, Tokyo, Japan) using 20× (UPlanFL N) dry, 60× (PlanApo N), and 100× (UPlanFL N) oil immersion objectives.

Processing Description

NanoSIMS instrument tuning and analysis:
Cell targets were identified (by SYBR stain) and marked on NanoSIMS membranes with a laser dissection microscope (LMD6000; Leica Microsystems) for ease of rediscovery on the NanoSIMS.  Samples were analyzed by raster ion imaging with a CAMECA NanoSIMS 50L at the Caltech Microanalysis Center in the Division of Geological and Planetary Sciences.  A focused primary Cs+ beam of ~ 1 pA was used for sample collection, with rasters of 256 × 256 or 512 × 512 pixels. 1H (EM#1), 2H (EM#2), 12C2 (EM#3), 13C12C (EM#4), 12C14N (EM#5), and 12C15N (EM#6) were measured simultaneously.Collection began after a pre-sputtering of equal intensity to one collection frame (~45 min). 

Data Processing:
Recorded images and data were processed using Look @ NanoSIMS software. Images were deadtime-corrected and individual ion image frames were merged and aligned using the 12C14N ion image to correct for drift during acquisition. Cell-based regions of interest (ROIs) were determined by "interactive thresholding" with the 12C14N ion image. Final ion images and counts per ROI were calculated by summation of ion counts for each pixel over all scans. Outputs for ROI size were used to compute cell diameters. We also confirmed that cell ROIs had a total C to total N ratio that was distinct from the background correction ROIs or coal to ensure drawn ROIs only included biomass targets.

Further information about the data output can be found in the LANS Manual (PDF).

BCO-DMO Data Processing:
- modified parameter names to conform with BCO-DMO naming conventions: changed % to pcnt; changed names of params starting with a number;
- replaced NaN and missing data with nd ("no data").

Instruments

Details
Instance Description (LMD6000)

Cell targets were identified (by SYBR stain) and marked on NanoSIMS membranes with a laser dissection microscope (LMD6000; Leica Microsystems) for ease of rediscovery on the NanoSIMS.

Instruments that generate enlarged images of samples using the phenomena of reflection and absorption of visible light. Includes conventional and inverted instruments. Also called a "light microscope".

Zeiss 1550 VP Field Emission Scanning Electron Microscope [Scanning Electron Microscope]
Details
Instance Description (Zeiss 1550 VP Field Emission Scanning Electron Microscope)

Scanning electron microscopy (SEM) of the filters was done on a Zeiss 1550 VP Field Emission Scanning Electron Microscope at the GPS Division Analytical Facility at Caltech.

Scanning electron microscope

BX51 epifluorescence microscope [Microscope-Fluorescence]
Details
Instance Description (BX51 epifluorescence microscope)

SYBR stained cells were imaged with a BX51 epifluorescence microscope (Olympus, Tokyo, Japan).

Instruments that generate enlarged images of samples using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption of visible light. Includes conventional and inverted instruments.

Parameters

file_name [file_name]
Details
file_name
Name of raw nanosims frame

File name

Sample [sample]
Details
Sample
Sample identifier

unique sample identification or number; any combination of alpha numeric characters; precise definition is file dependent

Condition [sample_descrip]
Details
Condition
Identifies amendments
text description of sample collected
Details
Morph
Morphology of the sample
text description of sample collected
Details
ROI
Region of interest; each file_name will have multiple regions of interest (ROIs), which is the single cell data
text description of sample collected
Xi [unknown]
Details
Xi

x-coordinate where the ROI is in the image frame

association with a community-wide standard parameter is not yet defined
Yi [unknown]
Details
Yi

y-coordinate where the ROI is in the image frame

association with a community-wide standard parameter is not yet defined
H1_MEANi [count]
Details
H1_MEANi
1H Mean ion counts
Number of individuals counted in sample or sample fraction
H1_Poiss_Ei [unknown]
Details
H1_Poiss_Ei
1H Poisson standard error
association with a community-wide standard parameter is not yet defined
H1_Poiss_pcnt_Ei [unknown]
Details
H1_Poiss_pcnt_Ei

1H Poisson percentage

association with a community-wide standard parameter is not yet defined
H1_SIZEi [unknown]
Details
H1_SIZEi

1H Size of ROI

association with a community-wide standard parameter is not yet defined
H1_PIXELSi [unknown]
Details
H1_PIXELSi
1H Number of pixels in ROI
association with a community-wide standard parameter is not yet defined
H1_LWratio [unknown]
Details
H1_LWratio
1H Length to width ratio of ROI
association with a community-wide standard parameter is not yet defined
H2_MEANi [count]
Details
H2_MEANi
2H Mean ion counts
Number of individuals counted in sample or sample fraction
H2_Poiss_Ei [unknown]
Details
H2_Poiss_Ei
2H Poisson standard error
association with a community-wide standard parameter is not yet defined
H2_Poiss_pcnt_Ei [unknown]
Details
H2_Poiss_pcnt_Ei

2H Poisson percentage

association with a community-wide standard parameter is not yet defined
H2_SIZEi [unknown]
Details
H2_SIZEi

2H Size of ROI

association with a community-wide standard parameter is not yet defined
H2_PIXELSi [unknown]
Details
H2_PIXELSi
2H Number of pixels in ROI
association with a community-wide standard parameter is not yet defined
H2_LWratio [unknown]
Details
H2_LWratio
2H Length to width ratio of ROI
association with a community-wide standard parameter is not yet defined
H2R_MEAN [unknown]
Details
H2R_MEAN
Mean ratio of minor ion to major ion
association with a community-wide standard parameter is not yet defined
H2f_MEAN [unknown]
Details
H2f_MEAN
Fractional abundance of minor ion to major ion
association with a community-wide standard parameter is not yet defined
C12_MEANi [count]
Details
C12_MEANi

12C2 Mean ion counts

Number of individuals counted in sample or sample fraction
C12_Poiss_Ei [unknown]
Details
C12_Poiss_Ei

12C2 Poisson standard error

association with a community-wide standard parameter is not yet defined
C12_Poiss_pcnt_Ei [unknown]
Details
C12_Poiss_pcnt_Ei

12C2 Poisson percentage

association with a community-wide standard parameter is not yet defined
C12_SIZEi [unknown]
Details
C12_SIZEi

12C2 Size of ROI

association with a community-wide standard parameter is not yet defined
C12_PIXELSi [unknown]
Details
C12_PIXELSi

12C2 Number of pixels in ROI

association with a community-wide standard parameter is not yet defined
C12_LWratio [unknown]
Details
C12_LWratio

12C2 Length to width ratio of ROI

association with a community-wide standard parameter is not yet defined
C13C12_MEANi [count]
Details
C13C12_MEANi

13C12C Mean ion counts

Number of individuals counted in sample or sample fraction
C13C12_Poiss_Ei [unknown]
Details
C13C12_Poiss_Ei

13C12C Poisson standard error

association with a community-wide standard parameter is not yet defined
C13C12_Poiss_pcnt_Ei [unknown]
Details
C13C12_Poiss_pcnt_Ei

13C12C Poisson percentage

association with a community-wide standard parameter is not yet defined
C13C12_SIZEi [unknown]
Details
C13C12_SIZEi

13C12C Size of ROI

association with a community-wide standard parameter is not yet defined
C13C12_PIXELSi [unknown]
Details
C13C12_PIXELSi

13C12C Number of pixels in ROI

association with a community-wide standard parameter is not yet defined
C13C12_LWratio [unknown]
Details
C13C12_LWratio

13C12C Length to width ratio of ROI

association with a community-wide standard parameter is not yet defined
C13R_MEAN [unknown]
Details
C13R_MEAN
mean ratio of minor ion to major ion
association with a community-wide standard parameter is not yet defined
C13f_MEAN [unknown]
Details
C13f_MEAN
fractional abundance of minor ion to major ion
association with a community-wide standard parameter is not yet defined
N14C12_MEANi [count]
Details
N14C12_MEANi

14N12C Mean ion counts

Number of individuals counted in sample or sample fraction
N14C12_Poiss_Ei [unknown]
Details
N14C12_Poiss_Ei

14N12C Poisson standard error

association with a community-wide standard parameter is not yet defined
N14C12_Poiss_pcnt_Ei [unknown]
Details
N14C12_Poiss_pcnt_Ei

14N12C Poisson percentage

association with a community-wide standard parameter is not yet defined
N14C12_SIZEi [unknown]
Details
N14C12_SIZEi

14N12C Size of ROI

association with a community-wide standard parameter is not yet defined
N14C12_PIXELSi [unknown]
Details
N14C12_PIXELSi

14N12C Number of pixels in ROI

association with a community-wide standard parameter is not yet defined
N14C12_LWratio [unknown]
Details
N14C12_LWratio

14N12C Length to width ratio of ROI

association with a community-wide standard parameter is not yet defined
N15C12_MEANi [unknown]
Details
N15C12_MEANi

15N12C Mean ion counts

association with a community-wide standard parameter is not yet defined
N15C12_Poiss_Ei [unknown]
Details
N15C12_Poiss_Ei

15N12C Poisson standard error

association with a community-wide standard parameter is not yet defined
N15C12_Poiss_pcnt_Ei [unknown]
Details
N15C12_Poiss_pcnt_Ei

15N12C Poisson percentage

association with a community-wide standard parameter is not yet defined
N15C12_SIZEi [unknown]
Details
N15C12_SIZEi

15N12C Size of ROI

association with a community-wide standard parameter is not yet defined
N15C12_PIXELSi [unknown]
Details
N15C12_PIXELSi

15N12C Number of pixels in ROI

association with a community-wide standard parameter is not yet defined
N15C12_LWratio [unknown]
Details
N15C12_LWratio

15N12C Length to width ratio of ROI

association with a community-wide standard parameter is not yet defined
N15R_MEAN [unknown]
Details
N15R_MEAN
mean ratio of minor ion to major ion
association with a community-wide standard parameter is not yet defined
N15f_MEAN [unknown]
Details
N15f_MEAN
fractional abundance of minor ion to major ion
association with a community-wide standard parameter is not yet defined
SumH [count]
Details
SumH
Sum of all ion counts across both ions of hydrogen
Number of individuals counted in sample or sample fraction
SumC [count]
Details
SumC
Sum of all ion counts across all ions of carbon
Number of individuals counted in sample or sample fraction
SumN [count]
Details
SumN
Sum of all ion counts across all ions of nitrogen
Number of individuals counted in sample or sample fraction

Dataset Maintainers

NameAffiliationContact
Elizabeth Trembath-ReichertCalifornia Institute of Technology (Caltech)
Shannon RauchWoods Hole Oceanographic Institution (WHOI BCO-DMO)

BCO-DMO Project Info

Project TitleDetermination of deep biosphere cell activity and identity utilizing the state of the art low-biomass, single cell techniques developed at JAMSTEC in their class 10,000 clean room
AcronymDeep biosphere cell activity
URLhttps://www.bco-dmo.org/project/672592
CreatedJanuary 6, 2017
ModifiedJanuary 6, 2017
Project Description

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.

Project Maintainers
NameAffiliationRoleContact
Elizabeth Trembath-ReichertCalifornia Institute of Technology (Caltech)Principal Investigator
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