Aerobic microbial life persists in oxic marine sediment as old as 101.5 million years

Materials

Sediment samples used in this study were collected by IODP Expedition 329 in the South Pacific Gyre (SPG; Supplementary Fig. 1)⁷. The samples collected from Site U1365 (23° 51.0377′S 165° 38.6502′W) are zeolitic and/or metalliferous pelagic clays, those from Site U1368 (27° 54.9920′S, 123° 09.6561′W) are calcareous nannofossil oozes, and those from Site U1370 (41° 51.1267′S, 153° 06.3674′W) are black metalliferous clay, containing light yellowish brown clay-bearing nannofossil ooze. The water depths of these sites are respectively 5697, 3739, and 5075 meters below sea level (mbsl) at U1365, U1368, and U1370. Whole-round core samples of U1365C 8H-2 (obtained from 68.9 meters below seafloor [mbsf]), 95.4 Ma), U1365C 9H-3 (74.5 mbsf), U1368D 1H-2 (1.6 mbsf), 1368D 2H-5 (14.7 mbsf), and U1370F 7H-6 (62.9 mbsf) were used for incubation experiments as described in the following section. Those samples were from horizons with no observable coring disturbance by visual core description³⁵. Drilling fluid contamination assessment by chemical tracer revealed minimal drilling contamination of the samples (≤1 cell/g-sediment for U1365C 8H-2 and 1368D 2H-5, below detection to 0 cell/g-sediment for U1365C 9H-3, U1368D 1H-2, and U1370F 7H-6)³⁵. All Expedition 329 data are archived and available online in the IODP database (http://iodp.tamu.edu/tasapps) and archived online in the IODP Expedition 329 Proceedings⁷. Additional sediment age estimation is available in reports by Dunlea et al.¹³ and Alvarez Zarikian et al.³⁶.

Incubation experiments

To identify autotrophic and heterotrophic microbial populations, as well as their potential rates for growth and substrate uptake, sediment samples were incubated with stable isotope-labeled substrates (¹³C₆-glucose, ¹³C₂-acetate, ¹³C₃-pyruvate, ¹³C-bicarbonate, ¹³C-¹⁵N-amino acids mix [mixture of 20 Amino Acids], and ¹⁵N-ammonium). The incubation experiments were initiated onboard during expedition. Avoiding outer edges of the sediment cores (which are likelier to be contaminated by drill fluid), each 15 cm³ sample was taken from an interior portion of a core with a sterile tip-cut 30 mL syringe, and placed in a 50 cm³ sterile glass vial (Nichidenrika-Glass Co. Ltd.) sealed with a sterile rubber stopper and a screw cap, followed by flushing with 0.22 μm filter-sterilized nitrogen gas and storage at 10 °C. All of the sterilization was done by autoclaving of the materials at 121 °C for 20 min. Given O₂ concentrations in interstitial water during Expedition 329 (~70 μM for U1365C 8H-2 and 9H-3, ~150 μM for U1368D 1H-2, ~130 μM for 1368D 2H-5, and ~1.5 μM for U1370F 7H-6⁷), we set O₂ concentration in the headspace of vials (other than U1370F 7H-6) at ~3.3% (v/v), which corresponds to the aqueous concentration of oxygen at ~43 μM (assuming salinity of sea water), by adding 0.22 μm filter-sterilized air. The labeled substrates were injected (15 μM of ¹³C-labeled substrates, and 1.5 μM of ¹⁵N-labeled ammonium, dissolved in 50–100 μL of sterile water) onto each subcore sample by syringe and needle and incubated at 10 °C (Supplementary Fig. 3a). All reagents and gas components, including air used for sample preparation, were filtered through 0.22 μm syringe top filter. After setting up the incubations, one of the vials from each set of incubation had no substrate added, and a sediment split from the same sample was fixed by adding equal volume (15 mL) of 4% paraformaldehyde (PFA) in PBS solution for 5 h at 4 °C (time point T0). At each of three time points (T1: ~3 weeks [21 days], T2: 6 weeks [68 days], T3: 18 months [557 days] after starting incubation), vials were opened and sediment samples were fixed with equal volume (15 mL) of 4% PFA in PBS solution for 5 h at 4 °C. Fixed samples were frozen at −80 °C. After storage, they were washed twice with PBS and preserved in PBS/ethanol (1:1 [v/v]) at −20 °C until analysis.

Cell enumeration and selective sorting onto the membrane

To efficiently analyze substrate incorporation into microbial cells with NanoSIMS, cells were separated from their sediment matrix and fluorescence-activated cell sorting (FACS) was conducted to concentrate and purify cells in a small area for analysis (~0.5 mm², Supplementary Fig. 3b)^14,37. Cell separation, enumeration, and FACS were all conducted in the clean-booth and clean-room facilities at the Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science and Technology (JAMSTEC).

Cell separation, microscopy, and sorting procedures followed the method of Morono et al.³⁷ with modifications. Eight milliliters of fixed slurry (1/3 [v/v] sediment in ethanol-PBS solution) was mixed with the same volume of 2.5% NaCl solution, followed by centrifugation at 4500 × g for 15 min, after which the supernatant was discarded and the pellet resuspended by adding 2.5% NaCl solution to be 20 mL of sediment slurry. The sediment slurry was added with 2.5 mL each of detergent mix³⁸ (100 mM EDTA, 100 mM sodium pyrophosphate, 1% [v/v] Tween 80) and methanol, then vigorously shaken for 60 min at 500 rpm using a Shake Master (Bio Medical Science, Tokyo, Japan). After shaking, the sediment slurry was sonicated (Bioruptor UCD-250; COSMO BIO) in an ice bath for 20 cycles of 30 s at 200 W on and 30 s off. The processed slurry was then carefully layered onto a manually layered high-density cushion solution consist of (from top) 4 mL of 30% [v/v] Nycodenz, 4 mL of 50% [v/v] nycodentz, 4 mL of 80% [v/v] Nycodentz, and 4 mL of 67% [w/v] of sodium polytungstate. Samples were centrifuged at 4000 × g for 120 min, after which the supernatant, including the high-density layer(s), was carefully removed and transferred to a separate vial. Cells in the supernatant were trapped onto an Anopore Inorganic Membrane (Anodisc, Whatmann, Kent, UK), washed with TE buffer, and then stained with 100 μL of SYBR Green I staining solution. After staining for 5 min, the SYBR-stained cells were washed with 2 mL of TE buffer, and then the membrane was placed into a 50 mL centrifuge tube containing 5 mL of TE buffer. Cells were detached from the membrane by sonication at 20 W for 10–30 s using a Model UH-50 Ultrasonic Homogenizer (SMT Co. Ltd., Tokyo, Japan) and concentrated to be 1.5 mL by centrifugation at 7000 × g for 10 min and discarding 3.5 mL of the supernatant. Part (0.5 mL) of the stained cell suspension was filtered onto 0.22-μm pore size black polycarbonate membrane (Isopore GTBP02500; Millipore) and used for counting microbial cells by the fluorescence color-based discriminative cell enumeration method^39,40. Cells were sorted following the flow cytometry protocol of Morono et al.³⁷ directly from the sorter onto NanoSIMS-compatible 0.2-μm polycarbonate filters coated with indium tin oxide (ITO)^14,41 and non-coated membrane (Isopore GTBP02500; Millipore). ITO coating on polycarbonate membranes (Isopore GTBP02500; Millipore) was prepared by a sputtering deposition technique at Astellatech Co. Ltd. The sorted cells on the non-coated membrane were stored at −20 °C until DNA extraction.

NanoSIMS analysis of single cell-image acquisition and data processing

Cell targets were identified by fluorescence of SYBR Green I stain and marked on NanoSIMS membranes with a laser dissection microscope (LMD6000; Leica Microsystems) for ease of rediscovery on the NanoSIMS (an example is shown in Supplementary Fig. 3b). Microbial cells that incorporated stable isotope-labeled substrates were analyzed using NanoSIMS 50L (AMETEK Co. Ltd., CAMECA BU) at the Kochi Institute for Core Sample Research, JAMSTEC. Samples on the ITO-coated polycarbonate membrane were pre-sputtered at high beam currents (30 pA/s/µm²) before measurement. The ¹²C⁻, ¹³C⁻, ¹²C¹⁴N⁻, ¹²C¹⁵N⁻ and ³²S⁻ secondary ions were collected and measured in parallel at a mass resolution of 8000 that is sufficient to separate ¹³C⁻ from the ¹²CH⁻ and ¹²C¹⁵N⁻ from ¹³C¹⁴N⁻. Samples were measured using a 1–2 pA Cs⁺ primary beam that was rastered over 25 × 25 µm field of a 256 × 256 pixels with a counting time of 5 ms per pixel. Recorded images and data were processed using CAMECA WinImage software and OpenMIMS plugin⁴² in ImageJ⁴³ distribution of Fiji⁴⁴. Different scans of each image were aligned to correct image drift during acquisition. Final images were created by adding the secondary ion counts of each recorded secondary ion from each pixel over all scans. Intracellular carbon and nitrogen uptake from stable isotope-labeled substrates was calculated by drawing regions of interest (ROI) on CN⁻ images (recognizing cells in the images) and calculating ¹³C/¹²C and the ¹⁵N/¹⁴N ratio (calculated from the ¹²C¹⁵N/¹²C¹⁴N ratio). Instrumental mass fractionation of NanoSIMS analysis was calibrated by the conversion factor obtained by comparing carbon and nitrogen isotopic ratios of E. coli cells of varying isotopic enrichments measured at single cells with NanoSIMS and at bulk with an elemental analyzer/isotope ratio mass spectrometer (EA/IRMS, FlashEA 1112/DeltaPlus Advantage, Thermo Fisher Scientific). Concentration of bicarbonate (DIC) in the original sample determined on board⁷ was used to calculate substrate incorporation ratio (atom %) for bicarbonate in single cells.

Biomass and isotope calculations

Isotope incorporation data analysis and display as violin plots of the kernel density function were done using R⁴⁵ with the “ggplot2”⁴⁶, “cowplot”⁴⁷, “ggsci”⁴⁸, “scales”⁴⁹ packages. The “active” cell ROIs, those incorporated ¹³C- and/or ¹⁵N-labeled substrates, were determined by their isotopic ratio exceeding the 99.7% confidence interval of background carbon and nitrogen isotopic abundance of polycarbonate membranes (1.24 atom % for ¹³C and 0.446 atom % for ¹⁵N). As measures for the rate of microbial biomass synthesis, we calculated the biomass-based specific growth rate and the substrate incorporation-based biomass generation rates. The biomass-based specific growth rates (µ_B, Eq. (1)) were calculated from the abundance of cells in the sediment samples at the start of incubation X₀ and the abundance at the incubation period t (Xt). The substrate incorporation-based biomass generation rates^14,50 (^Cµ_S and ^Nµ_S, Eqs. (2) and (3), for carbon and nitrogen substrates, respectively) were calculated from the fractional abundance of isotope label in cellular biomass, where μ_S is the biomass generation rate (encompassing both cell maintenance and generation of new cells), t is the length of the incubation, F_label is the labeling strength, F_t is the single-cell NanoSIMS measurement, and F_nat is the natural abundance. For the calculation of ^Cµ_S and ^Nµ_S, a conservative approach was used by only including ROIs where either ¹³C or ¹⁵N ratio was above the 99.7% confidence interval of backgrounds shown above. These rate calculations assumed that the carbon and nitrogen for the biomass generation were all derived from the substrates.

To document the physiological status of microorganisms in the original sediment samples, the fraction of microbes that originally existed in the sediment samples (f₀) was calculated from the observed active ROI fractional ratio (f_t), a factor of biomass increase (A) by following equation (Eqs. (4)–(6)).

If the number of ROIs was not enough to give enough sampling depth for fulfilling the Eq. (5), the f₀ could not be calculated and was shown as N.A.

DNA extraction and sequencing

Microbial cells on non-coated polycarbonate membrane were lysed using a protocol modified after Morono et al.⁵¹ at ISO Class 1 clean air environment in super-clean room of JAMSTEC-KOCHI⁵². Sorted cells on the membrane were lysed by three-step alkaline treatments to minimize fragmentation of extracted DNA. First, five microliters of denaturation buffer (200 mM KOH, 20 µM EDTA) was added to the tiny piece of membrane with sorted cells, incubated at room temperature for 5 min, followed by adding 5 μL of neutralization buffer (200 mM HCl, 300 mM Tris-HCl [pH7.5]). The liquid part was recovered for the second step, which was adding another 5 microliters of denaturation buffer, heating at 70 °C for 5 min, and adding 5 μL of neutralization buffer, followed by recovery of the liquid part and pooling with the first extract. The third step was adding an additional 5 μL of denaturation buffer, heating at 70 °C for 10 min, and adding 5 μL of neutralization buffer, followed by recovery of the liquid part. Heating was done with an Applied Biosystems Veriti Thermal Cycler (Thermo Fisher Scientific). The lysis of cells was confirmed by checking processed membrane with fluorescence microscope after staining with SYBR Green I (Thermo Fisher Scientific). Negative control extractions (8 separate extractions) were performed by using polycarbonate membrane without sorted cells. The resultant DNA extract was then used directly for first-round PCR amplification with universal primers targeting the V4 region of the 16S rRNA gene (U515F: 5′-TGY CAG CMG CCG CGG TAA-3′, U806R: 5′-GGA CTA CHV GGG TWT CTA AT-3′). Also, PCR No-Template-Control reactions (PCR-NTCs) run without template DNA (6 separate reactions) were performed. First-round PCR amplicons were quantified and purified via gel electrophoresis and then indexed and barcoded in the second PCR. The DNA sequencing was performed on a MiSeq Illumina platform following the methods outlined by Hoshino and Inagaki⁵³. The obtained sequence reads were quality trimmed and filtered by FASTQ Toolkit in Basespase (Illumina). Resultant paired-end reads were processed using mothur⁵⁴ following the Mothur software package (v.1.35.0) of Illumina MiSeq Standard Operating Procedure⁵⁵. All sequence reads from negative control extractions and PCR-NTCs, along with sequences from all samples were used to generate operational taxonomic units (OTUs) at 97% similarity using usearch 10.0 program⁵⁶. To remove sequences that may be of exogenous origin⁵⁷, the most conservative way to remove contaminant sequences considers all taxa that are identified in the laboratory controls as contaminants. This approach has the advantage of eliminating any false-positive discoveries of novel taxa; however, it has the disadvantage of introducing false-negative taxa, which may be of interest. For example, microbial species that are ubiquitous in environments may be excluded from a sample. To circumvent this issue, we employed a “probabilistic” approach to examining the microbial composition based on the consistency (i.e., variance) of the relative distribution of the taxonomic assignments or OTUs across the entire set of samples⁴¹. This approach compares the proportion of OTUs present between negative controls (i.e., extracted negative controls or PCR-NTCs) and samples, and determines the significantly more abundant OTUs in negative controls (ANOVA analysis) to be identified as contaminants and excluded from the sequence assemblage. By accounting for the consistency (variance) at which any taxon or OTU is found in the negative controls or set of experimental samples, a more nuanced view of the overall data set can be achieved. For the ANOVA analysis, we used Qiime⁵⁸ script group_significance.py to estimate a likelihood based on observed abundances for conditions (negative controls and experimental samples). This process was conducted sequentially on extraction negative controls and PCR-NTCs, and the OTUs that have significantly higher (p < 0.05, ANOVA) abundance in the extraction negative controls and the NTCs than in the incubation samples were identified and removed from further analysis. This read set was taxonomically classified by using the mothur utility package with the SSURef_NR99_123_SILVA database^59,60. The total sequences and relative abundance of taxa in the final sequence dataset are provided in Supplementary Data Set 2. The relative abundance of bacterial and archaeal OTUs at the genus level were exposed to non-metric multidimensional scaling with the metaMDS function of R software package vegan⁶¹. A Bray–Curtis distance matrix was used as dissimilarity index.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Source: Ecology - nature.com