Growth, morphology and depth-related changes

The Joetsu Basin dolomitic aggregates are easily distinguished from other minerals by their size, with diameters ranging from ~10 μm to ~150 μm (avg. diam. = 40 μm), and their distinctive morphologies e.g. “dumbbell pairs”, “chains”, or branching “cauliflower growths” (Fig. 3a–d). These distinctive morphologies can be seen to be formed from conjoined microdolomite spheres. Dark-coloured material is present at the cores of the microdolomites, and broken chains reveal interconnected internal porous regions (Fig. 3e,f). Macroscopically, the microdolomite appears as a very fine white or light-yellow powder, as the conjoined growth-patterns rarely exceed several grains and are sufficiently dispersed within the hydrate matrix that spheroidal aggregates do not merge to form crusts, veins, or other larger cemented mineralisations (Fig. 2c).

Figure 3 Mineral aggregates recovered from dissociated hydrate are relatively pure dolomite. (a) Light microscopy of single-grained dolomites showing dark inclusions (UTCW J25R, 53.9 mbsf, Mg/Ca = 0.91). (b) Single and paired “dumbbell” grains, showing layering in the internal dark portions (UTCW J22R, 28.7 mbsf, Mg:Ca = 0.92. (c) Shallow dumbbell grain (UTCW J21R, 12.2 mbsf, Mg:Ca = 0.74). Shallow grains (<20mbsf) show rough surfaces comprised of ~5 μm dolomite rhombs and low Mg/Ca ratios. (d) Deeper grains consist of smooth intergrown dolomite plates ~15 μm. The overall size of the deep grains ranges from 20 μm to > 150 μm and Mg:Ca ratios approaching 1 (UTCW J25R, 57 mbsf, Mg:Ca = 0.97). (e) Broken chain structure (UTCW J25R, 67.4 mbsf) shows smooth intergrowth of dolomite rhombs on the outer surface. (f) Close-up of previous grain showing concentric porous rings on the inside of the broken surface, possibly consisting of organic matter or residual fluid. Full size image

Although individual dolomite samples show some variation in the abundance of single grains, dumbbells, or cauliflower-shaped aggregates, the abundance of these morphologies does not change with depth. There is, however, an observable depth-related change in the outer texture and size of the aggregates. Hydrates sampled from <20 metres below seafloor (mbsf) contain aggregates of rough spheroidal angular dolomite rhombs (Fig. 3c). The rhombs are ~5 μm across, and form spheroidal aggregates ~15–20 μm in diameter. There is a transition to smoother surface textures between 20 mbsf and 30 mbsf, such that dolomite rhombs sampled at >30 mbsf have intergrown dolomite surfaces (Fig. 3d), comprising hexagonal plates or shield shapes ~10–15 μm across organized in spheroidal aggregates ranging from ~30–150 μm in diameter.

Non-biogenic microdolomite aggregates with similar spheroidal and dumbbell morphologies have been produced in the laboratory at temperatures of >40 °C through direct precipitation from a gel of magnesium-rich amorphous calcium carbonate (MgACC) which quickly transforms to spheroidal proto-dolomite and eventually undergoes dewatering to produce microdolomite17,18. During this transformation, Mg:Ca ratios increase from 0.65 in pure MgACC to 1.00 in stoichiometric dolomite, where Mg:Ca is the molar ratio of the two corresponding elements in the dolomite. In order to see if the Joetsu Basin dolomites show any systematic change, Mg:Ca was determined through Rietveld refinement of the x-ray diffraction patterns19 and by applying the equation of Turpin et al.20. Grain diameter was determined through microscopy21 (Supplementary Tables S1, S2). In general, both the grain size and average diameter of grains increase with depth (Fig. 4a). The smallest average grain diameter is 19 µm (UTNE at 16mbsf) while the largest average is 114 µm (UTCW at 88mbsf). At 22 µm, the Joetsu Knoll sample (JK at 30mbsf) is also quite small despite being much deeper than other small dolomite aggregates. The lowest Mg:Ca ratio is 0.76 (UTCW 12mbsf), while the highest is 0.99 (UTCW 86mbsf). There are, however some dolomites with Mg:Ca > 0.95 at depths as shallow as 20mbsf.

Figure 4 Depth profiles for recovered dolomite, hydrate gases, and oils found in hydrate. Point size is scaled to mean diameter of microdolomites (smallest at 19 µm and largest at 114 µm). Red = UTCW, blue = UTNE, yellow = JK. (a–c) The Mg:Ca ratios and stable isotopic composition of the dolomite aggregates. For δ18O, black line represents equilibrium values with seawater based on the geotherm. (d) Comparison of stable isotopic composition for Joetsu Basin microdolomites in this study (DOL) versus previously published MDACS from Umitaka Spur (upward triangles: Zhang et al.9; downward triangles:Hiruta et al.8). (e–g) Gas composition and stable isotopic composition for dissociated methane hydrate. The Δ13C values show the difference between dolomite and the hydrate CO 2 , illustrating the degree of disequilibrium that exists in shallow samples. Equilibrium values (solid line) based on the geotherm. (h–j) Organic chemistry of UTCW hydrate oil showing relative increases in refractory asphaltene and increases in C 29 25-norhopane at the expense of C 30 -hopane due to biodegradation. Full size image

Stable isotopic composition

Stable carbon isotope ratios can potentially indicate the carbon source of the microdolomites, particularly in the Joetsu Basin where the primary carbon pools have distinct isotopic compositions. The δ13C values for the microdolomites (Fig. 4b,d) are all positive, which is significantly different from the negative values associated with MDACS in the area8,9. Generally, dolomites which have positive δ13C values are the result of a carbon source related to methanogenesis which has subsequently undergone evaporation22. The Joetsu Basin hydrates have reported δ13C values ranging from −57.1‰ to −43.9‰VPDB in Joetsu Knoll and −36.6‰ to −34.6‰VPDB in Umitaka Spur5, both locations indicating a thermogenic source with some admixture of biogenic methane. Similarly, the δ13C values of methane reported for deep wells within the nearby Niigata gas fields range from −35‰ to −33‰VPDB23,24, overlapping with the range of values from Umitaka Spur. The reported δ13C values for dissolved inorganic carbon (DIC) in the interstitial waters of Joetsu Basin sediments range from +18.7‰ to +28.5‰VPDB at Joetsu Knoll and −4.9‰ to +41.4‰ at UTCW15; at both areas, the least positive values are shallow sediments near the sulphate methane transition (SMT), due to anaerobic oxidation of methane (AOM) whereas the most positive values are in the deeper sediments. Presumably the source of this DIC, which is enriched in 13C relative to 12C, is a deep source of residual organic that has undergone methanogenesis over long periods of time3,4. Porewaters with negative δ13C values for DIC are related to a combination of gas hydrate dissociation and AOM8,24.

During growth gas hydrate incorporates pore fluids resulting in residual porewaters with negative δ18O values12,13,25. The removal of porewater and the resulting dehydration is observed as salinity anomalies within interstitial water throughout surrounding hydrate locales of the Joetsu Basin11,14,15. Japan Sea bottom-waters in the Joetsu Basin are cold at 0.4 °C and the geothermal gradient at Umitaka Spur and Joetsu Knoll is 105 mK/m26. The temperature of the deepest samples at 90 mbsf, which is just above the gas hydrate stability zone (GHSZ) would be expected to be 9.9 °C. The δ18O value for dolomite in equilibrium with seawater down to this depth would range from +6.4‰ to +4.1‰VPDB27 (Supplementary Table S2). The dolomites show δ18O values to the left of equilibrium with seawater plotted as a solid line (Fig. 4c) due to isotopic fractionation between oxygen in the interstitial water and water-bound oxygen in the growing hydrate13,28. Both UTNE and JK dolomites show a greater degree of depletion of 18O than UTCW. If the extent of disequilibrium is taken as an indicator of the rate of growth, then most of the rapid growth of hydrates occurred at depths less than 20 mbsf. As hydrate is buried, the values again approach those of thermal equilibrium with seawater. Unlike the δ13C values, the δ18O values of MDACs in the UTCW area show considerable overlap with those values observed for the microdolomites (Fig. 4d). The larger, deeper microdolomites show less positive values, particularly at UTNE, indicating that hydrate growth at depth continues and in doing so takes up water from the fluid inclusions in which the microdolomites also continue to grow.

Non-hydrocarbon gases incorporated in hydrates

The Joetsu Basin hydrates contain both hydrogen sulphide and carbon dioxide5,7. Significant amounts of H 2 S (up to 10.8 mL/L-CH 4 ) were found in hydrates between 10 mbsf and 20 mbsf. These depths coincide with the formation of small dolomite grains (Fig. 4e) and it may be that the presence of high sulphide concentrations in the hydrate contributed to the initial stages of microdolomite precipitation29. Carbon dioxide is also present, and the δ13C values for CO 2 reach minimum values over the same interval (Fig. 4f) then gradually become more positive with depth as they reach equilibrium with the surrounding DIC-pool. The composition of non-hydrocarbon gases within the hydrate therefore seems to be influenced by AOM and sulphate reduction to some degree5, but only significantly so at depths of less than 20 mbsf. This is not the case for the microdolomites, as the δ13C of the UTNE microdolomites seems only to be influenced by AOM at shallow depths and not at all at UTCW (Fig. 4b). Assuming the aforementioned temperature gradient, we calculated Δ13C dolomite-CO2 as a function of depth (Supplementary Table S2)30. Calculating the equilibrium values for the microdolomites indicates a high degree of isotopic disequilibrium with the CO 2 for the hydrates from both UTNE and UTCW at <20 mbsf (Fig. 4g). Even though AOM may influence the δ13C of CO 2 in the hydrate, the primary source of carbon in the microdolomites must be 13C-enriched DIC, which can ultimately only derive from methanogenesis at greater depths or from some form of microbial activity which also produces CO 2 within saline fluid inclusions in the gas hydrate.

Oils, brines, and other impurities excluded during hydrate growth

It has been shown that rapid hydrate growth, at least in the case of synthetic hydrates, can lead to the temporary formation of encapsulated pockets of brine or finely dispersed saline fluid inclusions31. The Joetsu Basin hydrates recovered from the UTCW sites have yellow hydrate which, when dissociated, yields yellow oil which is emulsified in the clear hydrate water and microdolomite which settles to the bottom (Fig. 2c). The recognition of distinct insoluble oil and water phases is important because when trapped in pockets and veins, water-in-oil emulsions both stabilize brines providing microbial habitats32 and could potentially serve as a spherical-template for the formation of mineral precipitates such as the spheroidal microdolomites. Evidence for microbial processes can be found in the chemical composition of the oils which exhibit alteration consistent with the subsurface degradation of petroleum33. Specifically, the higher carbon-number n-alkanes and steranes which are generally present in undegraded oil are notably absent, and instead biodegradation-products such as the 25-norhopanes have been formed from regular hopanes, and the oils relatively enriched in recalcitrant components such as asphaltene (Supplementary Fig. S3 and Supplementary Table S4). The proportion of asphaltene, here taken as a more refractory organic component, becomes greater relative to total extractible organic matter (EOM) with depth, indicating that the biodegradation of the labile compounds is ongoing during burial especially in the upper 30mbsf (Fig. 4h) and is generally accompanied by an increase in the diameter of the microdolomite grains. Similarly, C 29 25-norhopane, which is formed directly from the biodegradation of C 30 hopane increases with depth (Fig. 4i) and the amount of C 30 hopane decreases relative to asphaltene.

Internal chemistry of dolomite grains

The majority of the microdolomites show zonation as do other microbial dolomites22; the Joetsu Basin microdolomites have outer rims with high Mg:Ca ratios and are optically clear while the darker central cores have lower Mg:Ca ratios, sometimes approaching Mg:Ca = 0.7 (Fig. 5). This may reflect that hydrate growth is initially rapid in shallow sediments and leads to less-ordered dolomites. However, during hydrate burial the dolomites grow slowly until the overall Mg:Ca ratio approaches 1 (Fig. 4a). Prior to analysis, and during the sample preparation, it was also noticed that some of the grains still contain fluid inside (Supplementary Fig. S2) which, if left to dry, formed secondary minerals on the surfaces of the polished microdolomites. EPMA showed that these grains have high Na and Cl in the cores, presumably trapped saline water.

Figure 5 (a) SEM scan of a composite “cauliflower” (Grain-A) and a single grain dolomite aggregate (Grain-B) from UTCW Site J25R (57.9mbsf) following elemental mapping. Blue line shows where quantitative elemental mapping was carried out at 2 μm intervals using EPMA. (b) EPMA mapping shows that areas high in Mg (green) are concentrated on the transparent outer surface, while areas to the center of the grains are relatively more enriched in Ca (red). (c) Quantitative scanning of Mg:Ca ratios across shows much lower Mg:Ca ratios near the dark cores and internal voids, particularly in the single-grain sample. (d) Transmission light microscopy shows concentric layering around dark core material. Full size image

Internal microbial content of dolomite grains

Epifluorescence microscopy of DNA-stained microdolomites indicated high concentrations of microbial DNA in two samples from shallow depths (less than 30 mbsf), and lesser concentrations in two samples beneath 30 mbsf. Despite differences in DNA-concentrations, both shallow and deep microdolomites could yield sufficient extractible DNA for 16S rRNA phylogenetic analysis (Supplementary Table S5). Epifluorescence microscopy showed that DNA lined the inner surfaces and cores of the spheres (Fig. 6), strongly indicating that phylogenetic information for the microdolomites pertains to their internal microhabitat.

Figure 6 (a) Epifluorescence imagery of dolomite grain showing internal presence of microbial DNA. In this case, the dolomite crystals forming the aggregate appear amber-colored while nucleic acid in the microbial material fluoresces due to SYBR-green dye (UTCW J24RB, 12.0 mbsf). (b) Fluorescence of the same sample (white box) showing high density of microbial DNA contained within surrounding microdolomite grains. (c) Pie charts showing the relative abundance of microbial phyla within deep and shallow samples. Full size image

It is notable that the microdolomites mostly lack sulphate reducing bacteria and ANME archaea that are associated with gas hydrate mounds and shallow sites of methane seepage34,35,36. A single sample (J20R 18.5 mbsf) had 0.5% archaea and 0.5% δ-proteobacteria (possible sulphate reducing bacteria), but aside from this the interior of the microdolomites appears to be a microhabitat distinct from that typically found at shallow sites of methane seepage. Instead, phylogenetic analysis suggests the microdolomites grew in a microhabitat similar to that hosted by deep gas hydrates37 and by marine oil spills38. For example, Sphingomonadales is present in all samples, and there is a notable predominance of α-proteobacteria in the deepest microdolomites (Rhizobiales makes up 50% of the microbial abundance in J25R 53.91 mbsf, compared to 17.9% for other α-proteobacteria in shallower samples). Both Sphingomonadales and Rhizobiales are reported oil-degraders.

However, there are differences to the communities reported from marine oil spills: β-proteobacteria, including Burkholderiales, which commonly occur in oxic-seawater near oil seep sites38 are absent, while γ-proteobacteria are of low abundance in shallow samples and absent in deeper samples. The low abundance of γ-proteobacteria and greater relative abundance of α-proteobacteria in the deepest samples is consistent with α-proteobacteria supplanting γ-proteobacteria during the later stages of petroleum degradation, after the lighter substrates have been removed39.

From the perspective of the formation of microdolomites, perhaps the most striking difference between the deep and shallow microbial communities is the abundance of Bacteroidetes; in particular Flavobacteriia which is present in the shallower samples (48.3% at 12.04 mbsf and 25.3% at 18.25 mbsf) and completely absent from the deeper samples. Because Flavobacteriia breaks down complex macromolecules including oils39, and produces extracellular polymeric substances that initiate the formation of spherulitic microdolomite40, Flavobacteriia likely plays a key role initiating the formation of microdolomites at shallow depths. Some strains of Flavobacteriia have a light-yellow colour39 and this may account for the yellow colour of some microdolomites and oils. Because both hydrate and sediment in Joetsu Basin samples contain oil, the sediments also contain some similar organotrophs41, as do sediments above deep gas hydrate on the Pacific side of Japan42, yet the formation of authigenic carbonates in the sediments appears to be predominantly a consequence of shallow ANME and SRB.

Of final note is the Phylum Firmicutes, including Class Bacilli, which is present in the shallow microdolomites, but more generally associated with hypersaline anoxic environments37. The Phylum Firmicutes is present at 11.8% in one shallow sample (J20R 18.25 mbsf), but the Class Bacilli is found only in trace amounts in both shallow samples and not at all in the deeper samples. In one of the deeper microdolomite samples (J25R 57.91 mbsf), the Phylum Firmicutes, Class Thermodesufovibrionia makes up 9.8% of the microbial distribution and its presence is most likely associated with the degradation of long-chain alkanes and fatty acids43.