Variations of organic functional chemistry in carbonaceous matter from the asteroid 162173 Ryugu

Domain: earth_science
Published: 2024-08-29
Editorial outcome: minor_revision

Abstract
Primordial carbon delivered to the early earth by asteroids and meteorites provided a diverse source of extraterrestrial organics from pre-existing simple organic compounds, complex solar-irradiated macromolecules, and macromolecules from extended hydrothermal processing. Surface regolith collected by the Hayabusa2 spacecraft from the carbon-rich asteroid 162173 Ryugu present a unique opportunity to untangle the sources and processing history of carbonaceous matter. Here we show carbonaceous grains in Ryugu can be classified into three main populations defined by spectral shape: Highly aromatic (HA), Alkyl-Aromatic (AA), and IOM-like (IL). These carbon populations may be related to primordial chemistry, since C and N isotopic compositions vary between the three groups. Diffuse carbon is occasionally dominated by molecular carbonate preferentially associated with coarse-grained phyllosilicate minerals. Compared to related carbonaceous meteorites, the greater diversity of organic functional chemistry in Ryugu indicate the pristine condition of these asteroid samples.

Introduction
The low-albedo asteroid 162173 Ryugu is one of many C-type asteroids1–3 that are thought to be parent bodies of carbonaceous chondrite meteorites that fall to Earth. Ryugu’s current form of a top-shaped, rubble-pile asteroid was created by a previous collisional disruption of a larger body3,4. However, orbital near-infrared spectroscopy detected a weak but ubiquitous 2.72 µm absorption feature indicative of hydrated minerals5, consistent with a pre-collisional history of hydrothermal aqueous processing. Preliminary examination of Hayabusa2 return samples by the JAXA curation team6,7 and the Hayabusa2 Initial Analysis Teams8,9 indicated petrographic, composition, and isotopic similarities to Ivuna-type (CI) carbonaceous chondrites. This group of rare chondritic meteorites are notable in that their bulk compositions closely match that of the solar photosphere, establishing a valuable reference material for the average isotopic and elemental composition of the early solar nebula. However, all CI chondrite samples show evidence of extensive aqueous alteration on their parent asteroid(s)10,11, and although the presence of extraterrestrial organic molecules has been demonstrated in these meteorites12–14, the question of how much of this alteration may be due to terrestrial contamination and weathering has not been resolved15–17.

The Hayabusa2 spacecraft collected surface regolith particles from Ryugu in two separate touchdown events, which were stored in collection chambers A and C of the spacecraft’s sample catcher18. These samples were protected within the Hayabusa2 re-entry capsule from atmospheric re-entry heating upon Earth arrival and have been stored under vacuum or an ultra-purified nitrogen atmosphere while at the JAXA curation facility. In this study, we investigate the functional group chemistry of discrete carbonaceous grains and diffuse carbonaceous matter within these particles. We analyzed three types of sample preparations: insoluble organic matter (IOM) residues extracted from particles by an HF/HCl dissolution procedure, thin sections of particle fragments prepared by ultramicrotomy, and thin sections extracted by focused ion beam (FIB) (Methods). A corresponding set of samples were also prepared from IOM and fragments of the CI meteorite Orgueil. While IOM residues provide a representative aggregated sampling of carbonaceous matter from each collection site on Ryugu, they are missing both soluble organic matter and valuable information regarding the local petrologic context with surrounding mineral grains. This missing context is provided by the ultramicrotome and FIB sections, although these preparations are less representative of Ryugu regolith. Comparisons between microtome and FIB sections are necessary to identify soluble organic matter and potential adverse effects of electron beam exposure to Ryugu carbonaceous phases from FIB.

The vast majority of the organic matter in carbonaceous chondrites is composed of a complex macromolecule made of interconnected polyaromatic moieties and aliphatic side chains19–21. X-ray absorption near-edge structure (XANES) spectroscopy is useful for characterizing such material, primarily because double- and triple-bonded carbon (e.g., C=C, C=O, C≡N) has large photoabsorption oscillator strengths for electronic transitions from core shell (1s) states to hybridized \pi* anti-bonding states22. Bulk XANES studies of IOM from many carbonaceous chondrites share common spectral features23, namely three peaks arising from aromatic C=C ring structures in polyaromatic domains, ketone (C=O) functional groups attached to aliphatic side chains, and carboxyl (COOH) functional groups attached to either aromatic or aliphatic carbons. Aliphatic carbons can also be detected by this method, although the oscillator strength for the corresponding 3p/\sigma* anti-bonding states is much weaker than their \pi* counterparts24. Thus, the presence of aliphatic features in carbon XANES spectra implies a high abundance of aliphatic carbon relative to \pi-bonded carbon groups. Small variations in the relative intensities of the main \pi* peaks observed between different meteorite group are consistent with the overall degree of alteration and heating of their respective asteroid parent bodies, with more aqueously altered chondrites containing decreasing ketone and carboxyl peaks, and heated chondrites containing increased aromatic peaks23,25. At the µm-to-sub-µm scale, the organic functional chemistry of IOM can be heterogeneous26. For example, some larger, discrete IOM grains occasionally show highly aromatic functional chemistries consistent with a higher concentration of slightly larger polyaromatic domains and shorter aliphatic side chains. Using analogous, higher spatial resolution methods in transmission electron microscopy (TEM), these functional group heterogeneities at scales down to a few nm have been observed within single carbonaceous grains27. Diffuse organic matter observed in situ in chondrite samples with XANES and TEM can show even more heterogeneous functional group compositions than those found in IOM samples25,28,29.

Here we apply these methods to assess the diversity of carbonaceous matter in Ryugu IOM samples A0106 and C0107 and intact particles from Ryugu aggregate samples A0108 and C0109. Correlated XANES, STEM, and nanoscale secondary ion mass spectrometry (NanoSIMS) of these samples reveals the morphology and chemical characteristics of different populations of carbonaceous material, including discrete carbon grains and diffuse carbonaceous matter (Supplementary Fig. 1). Shape analysis of XANES spectra using Gaussian fitting and a hierarchical clustering algorithm found three distinct functional chemistry populations. In addition, a unique molecular carbonate phase is present in FIB-prepared sections intermixed with coarse-grained phyllosilicates. Carbonaceous matter in both more abundant and more diverse than that in the CI chondrite Orgueil, indicating that asteroidal material is more pristine than its meteoritic counterparts on earth and holds promise to reveal new information about components and processes from the early solar system.

Results
IOM samples prepared from both Ryugu collection chambers show XANES characteristics similar to IOM from aqueously altered chondrite meteorites. “Bulk” spectra are similar to those of CI and CM (Mighei type) chondrites, both of which are among the most aqueously altered meteorite groups. However, a significant number of discrete, compact, grains have XANES spectra that deviate from bulk spectra (Fig. 1). We used spectral decomposition with a series of Gaussian peaks and a hierarchical clustering algorithm to perform a shape analysis and identify similarities within the spectral dataset (Methods; Supplementary Fig. 2). Shape analysis of XANES spectra from 23 discrete grains (Fig. 1D) indicate that twelve (about 50%) have IOM-like (IL) spectra, containing the three typical peaks, with most spectra showing slightly sharper ketone and carboxyl peaks (Fig. 1E). This slight difference in C=O features between bulk IOM and individual discrete grains was seen previously for nanoglobules within chondritic IOM residues26. Another seven (~30%) of these IOM grains show highly-aromatic (HA) spectra with a characteristic broadening of the aromatic C=C peak and minimal ketone and carboxyl peaks. Heirarchical clustering identified a group of four (~20%) Ryugu IOM grains distinct from the typical IL and HA spectral shapes. It consists of an intense but narrow aromatic peak and a subtle bump in the 287–288 eV range (Fig. 1E and Supplementary Fig. 3). The narrow aromatic peak centered at 285.0 eV indicates a more limited distribution of aromatic functionality, most likely an increase in the amount of single aromatic rings and small polyaromatic domains, as opposed to the larger polyaromatic moieties and broader aromatic peak observed in the highly aromatic grains. The greater 287–288 eV absorption, however, could be caused by a greater abundance of aromatic ring-bound carboxyl functional groups, amide groups, or aliphatic moieties22,24. Our finding that the XANES spectra from discrete grains cluster into three main spectral shapes matches the preliminary results from the Hayabusa2 Initial Analysis, where the AA spectral shape is identified in that study as aromatic8. Almost all N-XANES spectra of carbonaceous grains from each of the three spectral shapes are flat or contain a featureless bump (Supplementary Fig. 4), indicating low N abundance, in keeping with previous observations of CI and CM IOM (where N/C is typically ~3 at.%30). A non-detection of nitrogen with XANES has been shown to have a typical upper bound of N/C < 2 at.%31. Two out of three total N-XANES spectra from HA grains contain clear N absorption edges, suggesting preservation of some N in polyaromatic heterocycles, as observed in N-bearing nanoglobules in other carbonaceous chondrites26. However, the lack of an observed increase in N absorption in this third spectral type over HA or IL spectra (Supplementary Fig. 4) suggests contribution from amide functionality is unlikely. O-XANES spectra of Ryugu IOM, IL grains, and this third spectral type show a similar intensity ratio between the 531 eV \pi* absorption from carbonyl C=O functional groups and the \sigma* absorption at 540 eV from C-O single bonds32 (Supplementary Fig. 4), suggesting relatively similar abundances of O composition. In contrast, HA grains show a smaller \pi*: \sigma* peak ratio due to a smaller abundance of O-bearing functional groups. Thus, we tentatively describe this new alkyl-aromatic (AA) material as consisting of an increased amount of smaller polyaromatic domains, predominantly single or double benzene rings, decorated predominantly by carboxyl functional groups. This molecular interpretation is similar to that of Ryugu IOM and IL grains but with a limited range of small polyaromatic domains and longer aliphatic chains connecting them. This population of alkyl-rich macromolecular material could be related to the abundant alkylated polycyclic aromatic hydrocarbons (such as alkylbenzene) detected in Ryugu samples33.

This diversity in organic functional chemistry is also observed in microtome and FIB sections from Ryugu particles (Fig. 2). Carbonaceous material is found amidst fine-grained matrix material in x-ray photoabsorption maps. Discrete, compact grains are observed, including some with nanoglobule morphologies. Shape analysis of C-XANES spectra from these grains (Supplementary Fig. 5) shows that they fall into the three previously-observed spectral shape categories described above for Ryugu IOM. Heirarchical cluster analysis of 38 discrete grains in these sections determined a HA:AA:IL spectral shape ratio of ~20:25:55, similar to that of the IOM samples. Plotting the spectral contributions from the major absorption features in these spectra show a clear distinction between HA and IL spectral shapes, as well as the related cluster of AA grains (Fig. 2E). The overlap between the AA and IL fields in this plot illuminate how similar these two spectral shapes are to the human observer, and other non-traditional representations of C-XANES spectra may be necessary to distinguish them (e.g., Supplementary Fig. 7). Considering that these in situ sections are a less representative sampling of Ryugu than the IOM residues, these data suggest little difference in the diversity of functional chemistry between discrete carbonaceous grains in intact Ryugu particles and Ryugu IOM. In contrast, C-XANES measurements from 18 discrete carbonaceous grains in ultramicrotome sections from Orgueil matrix particles did not reveal any HA or AA spectral shapes, but only produced spectra consistent with IL functional chemistry (Supplementary Fig. 6), consistent with a previous XANES study of nanoglobules in Orgueil IOM26, although the low counting statistics (N=4) in that study cannot rule out the presence of grains with HA or AA spectral shapes in Orgueil.

Correlated STXM, STEM, and NanoSIMS-based N and C isotopic measurements of carbonaceous grains in a FIB liftout lamella extracted from Chamber A particle A0108-3 may suggest a connection between their functional chemistry and isotopic composition. Previous studies of Ryugu samples have indicated a large spread of 15N, 13C, and D values, but not correlated with functional chemistry of the organic matter. Here we report C-XANES data of 15 relatively large carbonaceous grains for which N and C isotopic compositions were previously measured (Fig. 3)8. Most of the measured carbonaceous grains in this section (small gray dots in Fig. 3E), identified by concentrations of carbonaceous matter in NanoSIMS image frames, have C and N isotopic compositions indistinguishable from the “bulk” isotopic composition of Ryugu organic matter8,34, within errors. However, most of the large carbonaceous grains deviate by >2\sigma in at least one of these isotopic systems (Supplementary Table 1). Both the highest 15N and 13C enrichments are observed in grains with an IL functional group chemistry, while the two measured grains with HA functional chemistry contain depletions in both 15N and 13C. Similar simultaneous large isotopic depletions in both 15N and 13C were previously observed in Ryugu carbonaceous grains8,35. In carbonaceous meteorite samples, correlated 15N and 13C depletions have been observed in carbonaceous grains within many primitive chondrites, including those that have experienced little aqueous alteration36,37 and other chondrites where aqueous alteration was more extensive38. While these data are very limited, they suggest that carbonaceous grains with different functional chemistry may compose distinct reservoirs of isotopic compositions, most likely reflecting distinct origins.

Diffuse organic matter is abundant in the fine-grained matrix of Ryugu8, visible in carbon STXM maps of microtome and FIB sections (e.g., Fig. 2B). The functional group chemistry of this diffuse organic matter varies from sample to sample but is usually consistent from region to region within a given section, suggesting a pervasive influence of parent body aqueous fluids. In previous XANES studies of CI chondrite meteorites, diffuse carbon is characterized by a dominant carboxyl functional group absorption, an absent or minor ketone peak, a minor amount of aromatic photoabsorption, and the occasional presence of a new peak at 290.4 eV assigned to CO3 functional groups25. Ryugu diffuse carbon is different in that most instances show a more intense aromatic peak and a clear ketone peak (Fig. 4). In all of the analyzed FIB sections in this study and one of the ultramicrotome sections, a 290.4 eV photoabsorption is also observed. XANES spectral mapping of this 290.4 eV peak in FIB and microtome sections reveals relatively large regions of diffuse organic matter (Fig. 4B), which is not similarly observed in CI or CM chondrites. For the FIB section from Ryugu sample A0108-3, this peak is accompanied by characteristic, strong, extended fine structure from carbonate minerals. This extended fine structure is present in a calcite grain in the section and within organic matter in the immediate surrounding mineral matrix, presumably taking the form of nanoscale calcite grains. Therefore, the 290.4 eV peak can confidently be assigned to CO3 functional groups in carbonate. In the others samples, however, no carbonate minerals were observed and no extended fine structure is present, suggesting this photoabsorption could be due to organic, molecular CO3. An abundant molecular carbonate phase was also previously reported in Ryugu matrix8,39. O-XANES spectra of this material lack a C=O peak, but this could be due to an overwhelming effect from the encompassing O-rich phyllosilicate groundmass.

Diffuse molecular CO3 in the Ryugu samples appear to be associated with phyllosilicate minerals instead of carbonates39. Spectral maps of the CO3 photoabsorption at 290.4 eV show a correlation between absorption and the distribution of phyllosilicates, with the greatest concentrations found where coarse-grained phyllosilicates were present, confirmed by subsequent high resolution STEM imaging (Fig. 4B, C). Other XANES studies of Ryugu organic matter also noted a distinct functional group composition in association with asteroidal phyllosilicates7,8, and their published spectra also contain a small CO3 peak at 290.4 eV. While previous XANES data from carbonaceous chondrite meteorites has established a spatial relationship between diffuse carbon and phyllosilicates arising from hydrothermal alteration of primary accreted amorphous silicate grains40,41, one TEM-based study of phyllosilicate minerals from the CI chondrites Orgueil and Ivuna, as well as the ungrouped carbonaceous chondrite Tagish Lake, also detected a strong CO3 energy-loss peak from isolated clay mineral grains42. This CO3 peak was not observed in carbonaceous aggregates with less developed phyllosilicate grains, leading the authors to conclude that coarse-grained clay minerals in CI chondrites may contain significant amounts of molecular CO3 bound to clay sheet surfaces and/or interlayer sites42. Our observations of the distribution of the XANES CO3 signal across the in situ Ryugu samples is consistent with this latter, clay-bound interpretation, and visual TEM confirmation of organic matter in between phyllosilicate sheets (Fig. 4C) indicates the presence of an abundant clay-associated polycarbonate material in Ryugu. While this spectral feature is prevalent in C-XANES data acquired from FIB sections, it is observed less frequently in ultramicrotome sections. The molecular CO3 signal appears preferentially spatially associated with clay mineral surfaces, but not prevalent in organic matter a few nm from the clays. The FIB samples have a relatively uniform thickness (~100 nm), providing much more clay volume, and hence more bound CO3 x-ray photoabsorption. In contrast, clay minerals in ultramicrotome sections have variable thickness, ranging from relatively isolated flakes only a few nm thick to phyllosilicate stacks up to 70 nm (i.e., the slice thickness). In addition, no molecular CO3 functionality was observed in Ryugu IOM samples, in which the clays and soluble organics have been removed.

Methods
IOM extraction
Residues of insoluble organic matter were prepared from Ryugu grains by acid demineralization after solvent-extraction of soluble organic matter. The samples were treated in a stirred 6N HCl solution with high-purity water in Teflon microtubes for ~48 h, followed by a 9N HF/1N HCl treatment for >48 h. This process was repeated three times, interspersed with 1N HCl rinses, high-purity water rinses, and a final methanol rinse. The final acid-insoluble, carbonaceous residues were transferred to glass vials and dried at 55°C on a hot plate. The glass vials were pre-heated to 500°C for 4.5 h in air prior to use. IOM samples of Murchison and Orgueil used in this study were sourced from Alexander et al.50.

Ultramicrotomy and focused ion beam preparation
Intact grains from Ryugu, meteorite analogs, and IOM powders were transferred with freshly-made glass needles and placed in molten droplets of sulfur kept at 114°C on a heated glass plate. Needle micromanipulation and monitoring/recording of this operation was done using an EXpressLO ex situ liftout system. Once the heat source was removed, the sulfur crystallized around the grains, and the entire droplet was attached to an 8mm epoxy block with a thin layer of cyanoacrylate adhesive. Ultramicrotomy was performed using a diamond knife in a Leica EM UC6 microtome. Sections were created at 70 nm thicknesses and placed on lacey carbon (for intact grains) or holey carbon (for IOM) support films on 200 mesh copper TEM grids for both STXM and TEM analysis.

Focused ion beam (FIB) liftout sections in this study were prepared at the U.S. Naval Research Laboratory with a Thermo Fisher Helios G3 DualBeam FIB and at the University of Tokyo with a Hitachi FB-2100 FIB. At NRL, Ryugu grains were transferred with freshly-made glass needles and deposited on double-sided copper sticky tape on a standard 12.5 mm pin mount. At UT, Ryugu grains were pressed into a 3mm gold disk with a diamond-stainless steel jig press. The gold disks were pre-cleaned with methanol/dichloromethane and Milli-Q water and then heated at 500°C for 3 h. Sample mounts were coated with ~25 nm of amorphous carbon or tungsten to minimize charging. Both FIB-SEM systems used 30 keV Ga+ ions to sputter away material, using typical FIB liftout protocols. with the exception that no electron imaging was performed on the lamellae after their thickness was less than 1.5 µm to avoid possible radiolysis damage to organic matter by the 5 keV electron beam51. Ion-assisted carbon deposition was used for the protective cover layer, which is easily recognizable with STXM and TEM. Lamellae were attached to copper half grids with ion-assisted platinum deposition before thinning to a final thickness of <100 nm. Once an adequate sample thickness was achieved, the lamellae were given a final cleaning step of 8–10 keV Ga+ ions on each side. Sections prepared at UT were further polished with a low energy Ar ion milling system (Fischione NanoMill 1040) at energies of 2 kV, 900 V, and 500 V.

Scanning transmission X-ray microscopy
STXM measurements were acquired at both beamline 19A at Photon Factory (PF), High Energy Accelerator Research Organization (KEK), Japan and beamline 5.3.2.2 at the Advanced Light Source, Berkeley, CA, USA. The synchrotron radiation X-ray source at compact STXM beamline 19A at PF employs an APPLE-II type undulator while ALS beamline 5.3.2.2 is illuminated by bending magnet radiation. The desired x-ray photon energies from this broadband source are selected with a monochromator grating and focused to a fixed ~35 nm beam spot using a Fresnel zone plate52. Prior to the experiments, the microscope energy control was calibrated at beamline 19A using highly ordered pyrolytic graphite and at beamline 5.3.2.2 using the sharp C and O photoabsorptions of CO2 gas introduced into the STXM chamber. The different calibration standards resulted in an energy shift of 0.2 eV between spectra acquired at the two facilities. During the experiments, the chambers for both beamlines were backfilled with He to ~30 kPa (0.3 bar) to provide temperature stability. Beamline 5.3.2.2 also includes a N gas filter, designed to block higher order interference from oxygen emission. This filter was used for initial measurements, but was removed after it was determined that nitrogen abundance was too low to affect carbon measurements. In hindsight, however, this choice is not optimal for in situ experiments (although it is valid for IOM samples), where organic grains could be closely associated with O-bearing silicate minerals, and some evidence for such interference was observed in subsequent Ryugu datasets, as a ubiquitous broad photoabsorption in C-XANES spectra.

X-ray absorption images were acquired at spatial resolutions up to 25 nm/pixel with a 2–3 ms dwell time per pixel, controlled by motion of the piezo stage motors. However, hyperspectral data “stacks” were acquired at 50 nm/pixel resolution to maximize signal-to-noise while minimizing total photon dose. Locations of discrete carbonaceous grains in these stacks are shown in Supplementary Fig. 1. In the near-edge structure region of the energy range, energy steps were set at 0.1 eV resolution for carbon stacks, and 0.2 eV for nitrogen and oxygen stacks. The optical density of each image in the data stack is calculated from background absorption (I0) by OD = −log(I/I0). XANES spectra of carbonaceous regions of interest are generated by summing pixels in those regions in the optical density datasets. PF STXM data were processed using the Python package Mantis53 and custom Python scripts8. Individual XANES spectra were deconvolved by fitting to a series of twenty Gaussian peaks of fixed position and width (allowing for peak height to vary)31, and a Python-based hierarchical, agglomerative clustering algorithm using the Ward variance minimization metric was applied to this parameterized, 20-dimensional dataset to identify clusters of similar spectral shapes (Fig. 1D and Supplementary Figs. 5, 6).

Scanning transmission electron microscopy
High angle annular dark field (HAADF) and bright field STEM images in this study were obtained with at the Naval Research Laboratory with a Nion UltraSTEM 200-X operated at 60 kV, with a nominal probe size of 0.15 nm and probe currents of 50–100 pA. Samples were baked at 140°C to maintain the ultra-high vacuum of the microscope.

Secondary ion mass spectrometry
The FIB section extracted from Ryugu grain A0108-3 was returned to the FIB, where it was removed from the TEM grid and welded with Pt flat onto a clean Au foil. A thin Au coating was applied to avoid surface charging effects, and the sample was analyzed with the Cameca NanoSIMS 50L at the Carnegie Institution of Washington. A focused Cs+ beam was rastered across the section with simultaneous collection of negative ions and secondary electrons. A large beam (hundreds of pA) was first used to pre-sputter the section to remove the Au coating and any surface contamination until stable secondary ion currents were obtained. A ~0.4 pA beam, at <100 nm diameter, was then rastered (256 × 256 pixels over a 10 µm × 10 µm region, 100 image frames of 65 s each) over the sample with simultaneous collection of negative secondary ions of 16O, 12C, 12C13C, 12C14N, 12C15N, 28Si, and 32S. The NanoSIMS ion images were analyzed with the L’image software (L. Nittler). After ion images were corrected for detector dead time and aligned, isotopic ratio images were generated and C-rich regions of interest were defined through a combination of automatic image segmentation and manual selection, based on C grains identified by the previous STXM and STEM investigations. Synthetic Si3N4 grains, as well as IOM from a CR chondrite, were used to correct the data for instrumental fractionation.

Data availability
STXM-XANES spectra reported in this paper were collected as part of the Hayabusa2 Initial Analysis Team activities, and have been deposited in the Data Archives and Transmission System (DARTS) at https://data.darts.isas.jaxa.jp/pub/hayabusa2/paper/sample/DeGregorio_2024/. Raw STXM data acquired at the Advanced Light Source are protected and are not available due to U.S. Department of Energy data privacy laws. However, raw STXM data from this study are available upon request from the beamline scientist (Matthew Marcus; mamarcus@lbl.gov).

Code availability
STXM datasets were processed using custom Python scripts developed and reported by Hikaru et al8. Spectrum shape analysis used the standard hierarchical clustering package from scipy.cluster.heirarchy, available in most Python implementations.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper reports on an excellent dataset from Ryugu organic matter (OM) acquired by a combination of XANES-STEM-NanoSIMS methods on extracted “insoluble OM” as well as on minimally processed Ryugu particles and their OM grains. The complementary information on functional chemistry and isotopic composition is of particular importance for this new type of extraterrestrial sample brought back from an asteroid. The authors claim that they may have located a new type of organic material (“alkyl-aromatic”, AA) within Ryugu OM, which is possibly characterized by a different functional chemistry, i.e., a narrow aromatic “285 eV” peak and a spectral “bump” in the 287-289 eV range due to more abundant carboxyl and aliphatic carbon bonding environments. If this is really the case, then this work is worth being published in Nature Communications, especially if the combination with isotopic C-N data indicates a different origin or evolution of this material.\nHowever, at the moment, I do not really see that this is the case. In Fig. 1, the four OM grains of the AA type look indeed very different to the “highly aromatic” (HA) grains, but I do not see a clear distinction between “IOM-like” (IL) and AA grains. The 285 band of the IL grains is also a bit narrower compared with the HA type, and in some of the IL spectra the carboxyl band is also not very sharp or distinct, but more like a “bump” (e.g., grains A2 or B2). This is also seen in their quantification ternary diagram (Fig. 2), where the AA and IL grains overlap. It is also known from meteorite OM investigations that the third IOM band (~288 eV, “carboxylic”) can be highly variable among different samples and even among the same meteorite in differently altered lithologies.\nFurthermore, although the authors state in the abstract that isotopic compositions vary between the three functional group types, I only recognize this in Fig. 3 for the HA and IL/AA grains, which seem to show this difference. The isotope data is based on a very limited dataset from one lamella, so if it is possible to verify that different types of OM bonding environments have different isotopic compositions, this would be also highly interesting.\nThey also argue that the OM types differ at the O K-edge, because the HA/AA grains show a different shape at 531 eV than the IL grains or bulk IOM. However, this figure is hidden in the supplementary file (Fig. S2), and if this information is important to underline this distinction it would be good to show in the main manuscript, maybe even with some further explanations.\nThey also state that “diffuse OM” in Ryugu samples might be more ubiquitous relative to meteorite samples (L. 388) to underline their assertion that Ryugu OM has sampled more pristine organics. However, I do not think that this is true, because it has been shown by several authors (Le Guillou, Vinogradoff, Changela, …) that this diffuse material is widespread in meteorites, but simply more difficult to study than the more globular OM grains, indicating a sampling bias.\nThere are already some functional group investigations on Ryugu OM by, e.g., Yabuta+, Ito+, and very recently by Stroud+ using very similar techniques, and in none of that work is there some indication for a new type of OM material with a different spectral shape. The only exception is the highly aliphatic material found by Ito+, but it is a bit surprising that the authors do not see strong indications of that band in their “AA” spectra, especially if they claim that the smaller aromatic units must be interconnected by longer aliphatic chains (L. 214).\nTo summarize, I would support publication of this paper in Nature Communication if the authors could strengthen their hypothesis that this new AA-material is really unique to Ryugu based on, for example, additional functional group analyses or quantifications (O-K, …) and/or more isotope data.", "reviewer_2": "The manuscript by De Gregorio et al. describes the results obtained from the analysis of various sample preparations of Ryugu material (IOM, thin sections of particle fragments prepared by ultramicrotomy, thin sections extracted by FIB) on carbonaceous matter. Different techniques were used for these characterizations: XANES spectroscopy, STEM, NanoSIMS. Three main results are discussed:\n1) the detection of carbonaceous grains belonging to 3 spectral types (from XANES data). Two correspond to groups identified in previous studies while the third one displays a singular XANES spectral shape, that the authors tentatively interpret to be due to an increased amount of smaller polyaromatic domains, predominantly single or double benzene rings, decorated predominantly by carboxyl functional groups (referred to as “alkyl-aromatic” (AA) material).\n2) A potential link between the functional chemistry of the 3 XANES spectral types identified and their isotopic composition, possibly reflecting distinct origins.\n3) The presence of CO3 functional groups in the diffuse carbonaceous material, associated with phyllosilicates, similar to CI/CM meteorites.\nThe manuscript is well written and the structure is clear. The results presented in the paper tend to extend previous analyses on Ryugu samples (e.g., Yabuta et al. 2023). The novelty of the results is, however, not always fully clear and would require some clarifications. The presentation of the results could also be improved in some cases, in particular for the XANES analyses. I detail my different comments below:\n- The description of the three XANES spectral types in the first part would require some clarifications. Some of the spectral differences described in the text are not clearly visible in Figure 1. While I understand why the spectra are shown with an offset, this makes it very difficult to assess possible differences between the spectral types. I would suggest to have an additional figure with spectra of the different types overplotted to make a proper comparison. I would also strongly suggest to add vertical lines to clearly see the abscissas of the different peaks. I would also strongly recommend to add arrows that indicate the features describes in the text.\n- To continue on my previous comment, looking at Figure 1E, I’m not convinced that the 3rd kind of spectra (AA), presented by the authors as novel, is really different from the “IOM-like”. I thus strongly encourage the authors to clarify/improve the Figure.\n- Figure 2: can you clarify why green and purple color fields overlap?\n- The authors claim that correlated STXM, STEM, and NanoSIMS data suggest that there is a link between the functional chemistry of the 3 XANES spectral types identified and their isotopic composition. As mentioned by the authors, the dataset is really limited, and the results shown in Figure 3 and highlighted in the text (“Both the highest 15N and 13C enrichments are observed in grains with an IL functional group chemistry, while the two measured grains with HA functional chemistry contain depletions in both 15N and 13C”) do not appear sufficient for such a claim. The large variations of 15N and 13C have already been observed in previous studies (e.g. Figure 6 of Yabuta et al. 2023) and discussed. It is not clear what is new here and what how does that lead to the conclusion that there is a link between the functional chemistry and isotopic composition.\n- Concerning the identification of CO3 rich material, it displays a 290.4eV peak assigned to CO3 functional groups, but lacks a C=O peak. In parallel, the authors rule out carbonate minerals as there is a lack of a characteristic, strong, extended fine structure typically observed in A0108-3 (Figure 4). Can you clarify if the latter (due to carbonate minerals) could be also “hidden”, similar to the C=O peak or because of a different process? In particular, such 290.4eV peak has also been observed by Yabuta et al. 2023. Similarly, no fine structure at higher energies (294 to 304 eV) could be observed, and Yabuta et al. concluded that the carbonates were not in a crystalline structure but more likely present as molecular carbonate. They also suggested that the Mg signature observed on clay-bound organics could indicate that Mg “be associated with molecular CO3, MgCO3, or a combination of these”.\n- “and visual TEM confirmation of organic matter in between phyllosilicate sheets (Figure 4D) indicates the presence of an abundant clay-associated polycarbonate material in Ryugu.” => I think Figure 4C should be referenced instead of Figure 4D.\n- Figure 4C. I would suggest to add some information on the image and the label to clarify what we observe on the image and why it indicates the presence of an abundant clay-associated polycarbonate material."}, "author_response": "Response to Reviewers – NCOMMS-24-03273A\nResponse to Reviewer #1:\nThis paper reports on an excellent dataset from Ryugu organic matter (OM) acquired by a combination of XANES-STEM-NanoSIMS methods on extracted “insoluble OM” as well as on minimally processed Ryugu particles and their OM grains. The complementary information on functional chemistry and isotopic composition is of particular importance for this new type of extraterrestrial sample brought back from an asteroid. The authors claim that they may have located a new type of organic material (“alkyl-aromatic”, AA) within Ryugu OM, which is possibly characterized by a different functional chemistry, i.e., a narrow aromatic “285 eV” peak and a spectral “bump” in the 287-289 eV range due to more abundant carboxyl and aliphatic carbon bonding environments. If this is really the case, then this work is worth being published in Nature Communications, especially if the combination with isotopic C-N data indicates a different origin or evolution of this material.\nHowever, at the moment, I do not really see that this is the case. In Fig. 1, the four OM grains of the AA type look indeed very different to the “highly aromatic” (HA) grains, but I do not see a clear distinction between “IOM-like” (IL) and AA grains. The 285 band of the IL grains is also a bit narrower compared with the HA type, and in some of the IL spectra the carboxyl band is also not very sharp or distinct, but more like a “bump” (e.g., grains A2 or B2). This is also seen in their quantification ternary diagram (Fig. 2), where the AA and IL grains overlap. It is also known from meteorite OM investigations that the third IOM band (~288 eV, “carboxylic”) can be highly variable among different samples and even among the same meteorite in differently altered lithologies.\nThe reviewer is correct in that the differences between AA and IL spectra are subtle. Except in the most obvious cases, most readers would not spot the differences. However, our hierarchical clustering algorithm consistently identified clusters with the AA spectral shape that are distinct from the IL spectral shapes. Both macromolecular structures are similar in that they are polyaromatic domains interconnected by aliphatic side chains, and modified by various functional groups. But the AA spectra are unique due to their intense and sharp 285 eV absorption and increase in aliphatic content.\nWe have softened our language regarding the distinction of these two spectral shapes throughout the paper. We have also removed all mentions of the AA shape being novel or new, as it has already been reported in Yabuta et al. (2023). The reviewer mentions the ternary diagram in Figure 2 where the color fields overlap for the IL and AA spectra, and we have added the following text to address this overlap:\n“Plotting the spectral contributions from the major absorption features in these spectra show a clear distinction between HA and IL spectral shapes, as well as the related cluster of AA grains (Figure 2E). The overlap between the AA and IL fields in this plot illuminate how similar these two spectral shapes are to the human observer, and other non-traditional representations of C-XANES spectra may be necessary to distinguish them (e.g., Suppl. Figure S7).”\nFigure S7 shows a comparison of peak height and peak width of the aromatic absorption at 285 eV, in which AA and IL spectra have separate fields.\nWe have also added more explicit mentions of our hierarchical clustering methods for identifiying spectral shape, to make clear that we are not relying on human identification of spectral shapes:\n“We used spectral decomposition with a series of Gaussian peaks and a hierarchical clustering algorithm to perform a shape analysis and identify similarities within the spectral dataset (Methods; Suppl. Figure S2).”\n“Heirarchical clustering identified a group of four (~20%) Ryugu IOM grains distinct from the typical IL and HA spectral shapes.”\nFinally, concerning the variability of the carboxyl band, we refer to the paper by Urquhart and Ade (2002), who provided a thorough investigation of the photoabsorption of the C=O functional group in various molecules. They found that there was a gap in the range between 286.7 and 288 eV, in both their experimental and ab initio results. While we cannot rule out a more complicated carbonyl-bearing functional group absorption in this energy region, we find it more likely that our interpretation of aliphatic photoabsorption in this energy region to be more likely.\nFurthermore, although the authors state in the abstract that isotopic compositions vary between the three functional group types, I only recognize this in Fig. 3 for the HA and IL/AA grains, which seem to show this difference. The isotope data is based on a very limited dataset from one lamella, so if it is possible to verify that different types of OM bonding environments have different isotopic compositions, this would be also highly interesting.\nIt is unfortunately that this is the only data available to us from the Hayabusa2 Initial Analysis period. We intend to continue collecting correlated NanoSIMS and C-XANES data on Ryugu samples, but those datasets will not be completed until Fall 2024. We have used careful language in our manuscript that the current data merely suggests the likelihood of a connection between functional chemistry and isotopic composition of carbonaceous grains in Ryugu, and have added a “may” in one instance to further soften our implication. We hope that the editors accept this response so as to not delay publication further.\nThey also argue that the OM types differ at the O K-edge, because the HA/AA grains show a different shape at 531 eV than the IL grains or bulk IOM. However, this figure is hidden in the supplementary file (Fig. S2), and if this information is important to underline this distinction it would be good to show in the main manuscript, maybe even with some further explanations.\nAfter careful reanalysis of our O-XANES data, we have concluded that the few spectra we have do not show enough of a distinction between the IL and AA spectral shapes. Most of our O-XANES data are acquired from microtome samples, for which contributions from the surrounding silicate minerals play an outsized role. Therefore we have removed the text describing differences in O-XANES spectra and replaced it with:\n“O-XANES spectra of Ryugu IOM, IL grains, and this third spectral type show a similar intensity ratio between the 531 eV π* absorption from carbonyl C=O functional groups and the σ* absorption at 540 eV from C-O single bonds34 (Suppl. Figure S4), suggesting relatively similar abundances of O composition. In contrast, HA grains show a smaller π*:σ* peak ratio due to a smaller abundance of O-bearing functional groups.”\nBecause the O-XANES data do not play a significant role in the discussion anymore, we have opted to keep this figure in the Supplementary Materials.\nThey also state that “diffuse OM” in Ryugu samples might be more ubiquitous relative to meteorite samples (L. 388) to underline their assertion that Ryugu OM has sampled more pristine organics. However, I do not think that this is true, because it has been shown by several authors (Le Guillou, Vinogradoff, Changela, …) that this diffuse material is widespread in meteorites, but simply more difficult to study than the more globular OM grains, indicating a sampling bias.\nWe disagree with this comment. Our data, and shown in Figure 4B, reveal that diffuse organic matter gives strong photoabsorption across all FIB and microtome sections, indicating a high abundance. We simply do not ever observe such high concentrations of diffuse organics in meteorites samples, and this is seen in the papers the reviewer mentions, where only small patches of diffuse organics are identified.\nTo support our conclusion, we have added the following text:\n“XANES spectral mapping of this 290.4 eV peak in FIB and microtome sections reveals relatively large regions of diffuse organic matter (Figure 4B), which is not similarly observed in CI or CM chondrites.”\nThere are already some functional group investigations on Ryugu OM by, e.g., Yabuta+, Ito+, and very recently by Stroud+ using very similar techniques, and in none of that work is there some indication for a new type of OM material with a different spectral shape. The only exception is the highly aliphatic material found by Ito+, but it is a bit surprising that the authors do not see strong indications of that band in their “AA” spectra, especially if they claim that the smaller aromatic units must be interconnected by longer aliphatic chains (L. 214).\nOur results are most similar to that of Yabuta et al. (2023), and that study describes two new types of organic matter based on XANES data. The first is called “aromatic”, which matches what we call “alkyl-aromatic” (or AA type) in our study. The second is called “molecular carbonate” by Yabuta et al., which we also identify in our dataset and also call “molecular carbonate”. We have added a sentence in the text to be more clear that we are not purporting to have discovered yet another type of organic matter in Ryugu:\n“Our finding that the XANES spectra from discrete grains cluster into three main spectral shapes matches the preliminary results from the Hayabusa2 Initial Analysis, where in that study the AA spectral shape is identified as “aromatic”14.\nThe data in Ito et al. (2022) is quite interesting. We were looking to find similar spectra in our samples, but we did not. I have analyzed that STXM data shown in Ito et al., and can reproduce their results. I have seen other presentations at conferences that suggest even one more additional aliphatic-rich spectral type as well. As researchers investigate more Ryugu samples, I believe we will see more published examples of exotic XANES spectral shapes. But that is outside the scope of our present study.\nTo summarize, I would support publication of this paper in Nature Communication if the authors could strengthen their hypothesis that this new AA-material is really unique to Ryugu based on, for example, additional functional group analyses or quantifications (O-K, …) and/or more isotope data.\nWe would like to clarify that although we find that AA organic matter appears to be unique to the Ryugu asteroid, we are not suggesting that it cannot exist in the related CI chondrites as well. Future work on CI chondrites may reveal AA grains that would not have been recognized due to their similarities to IL spectral shapes. We have added to the following text:\n“Application of automated spectral shape analysis to future studies of organic matter in CI chondrites may also discover examples of AA grains in these Ryugu analog samples.”\nWe believe that the changes we have made to the text and figures are sufficient to merit publication in Nature Communications.\nResponse to Reviewer #2:\nThe manuscript by De Gregorio et al. describes the results obtained from the analysis of various sample preparations of Ryugu material (IOM, thin sections of particle fragments prepared by ultramicrotomy, thin sections extracted by FIB) on carbonaceous matter. Different techniques were used for these characterizations: XANES spectroscopy, STEM, NanoSIMS. Three main results are discussed:\n1) the detection of carbonaceous grains belonging to 3 spectral types (from XANES data). Two correspond to groups identified in previous studies while the third one displays a singular XANES spectral shape, that the authors tentatively interpret to be due to an increased amount of smaller polyaromatic domains, predominantly single or double benzene rings, decorated predominantly by carboxyl functional groups (referred to as “alkyl-aromatic” (AA) material).\n2) A potential link between the functional chemistry of the 3 XANES spectral types identified and their isotopic composition, possibly reflecting distinct origins.\n3) The presence of CO3 functional groups in the diffuse carbonaceous material, associated with phyllosilicates, similar to CI/CM meteorites.\nThe manuscript is well written and the structure is clear. The results presented in the paper tend to extend previous analyses on Ryugu samples (e.g., Yabuta et al. 2023). The novelty of the results is, however, not always fully clear and would require some clarifications. The presentation of the results could also be improved in some cases, in particular for the XANES analyses. I detail my different comments below:\nWe thank the reviewer for their comments. Our study is indeed an extension of the work by Yabuta et al. (2023), for which many of the authors of the present paper also contributed. The present study includes new data, which supports the original conclusions of Yabuta et al. (2023).\n- The description of the three XANES spectral types in the first part would require some clarifications. Some of the spectral differences described in the text are not clearly visible in Figure 1. While I understand why the spectra are shown with an offset, this makes it very difficult to assess possible differences between the spectral types. I would suggest to have an additional figure with spectra of the different types overplotted to make a proper comparison. I would also strongly suggest to add vertical lines to clearly see the abscissas of the different peaks. I would also strongly recommend to add arrows that indicate the features describes in the text.\nWe have added lines to Figure 1E to denote the locations of the photoabsorptions for aromatic carbon (C=C), ketone (C=O), and carboxyl (COOH) functional groups. Text annotations of these lines identify them without the need to add arrows. We have also added a new figure to the supplementary information showing overlapping spectra from each of the three main spectral types, along with a plot of these spectra differences to Ryugu IOM, showing where each spectral types have more or less absorption relative to IOM.\nText additions related to this comment about the similarities between IL and AA spectral shapes can be found in our responses to the first reviewer.\n- To continue on my previous comment, looking at Figure 1E, I’m not convinced that the 3rd kind of spectra (AA), presented by the authors as novel, is really different from the “IOM-like”. I thus strongly encourage the authors to clarify/improve the Figure.\nThis comment is an extension of the previous comment. To address this, we have added a new figure to the supplementary information showing spectral differences between the different spectral shapes. In this plot, there is excess intensity (x-ray absorption) for the alkyl-aromatic spectral shape between 287-288 eV.\n- Figure 2: can you clarify why green and purple color fields overlap?\nThis comment was also pointed out by the first reviewer and has been addressed by our previous response to said reviewer.\n- The authors claim that correlated STXM, STEM, and NanoSIMS data suggest that there is a link between the functional chemistry of the 3 XANES spectral types identified and their isotopic composition. As mentioned by the authors, the dataset is really limited, and the results shown in Figure 3 and highlighted in the text (“Both the highest 15N and 13C enrichments are observed in grains with an IL functional group chemistry, while the two measured grains with HA functional chemistry contain depletions in both 15N and 13C”) do not appear sufficient for such a claim. The large variations of 15N and 13C have already been observed in previous studies (e.g. Figure 6 of Yabuta et al. 2023) and discussed. It is not clear what is new here and what how does that lead to the conclusion that there is a link between the functional chemistry and isotopic composition.\nWe believe the language we use in this paper establishes that we are merely reporting a possible connection between functional chemistry and isotopic composition. We have added text to this paragraph to clarify that we are tying the C-XANES data (new) to existing NanoSIMS data, emphasizing what is new in our study:\n“Previous studies of Ryugu samples have indicated a large spread of 15N, 13C, and D values, but not correlated with functional chemistry of the organic matter. Here we report C-XANES data of 15 relatively large carbonaceous grains for which N and C isotopic compositions were previously measured (Figure 3) 14,. Most of the measured carbonaceous grains in this section (small gray dots in Figure 3E), identified by concentrations of carbonaceous matter in NanoSIMS image frames, have C and N isotopic compositions indistinguishable from the “bulk” isotopic composition of Ryugu organic matter14,36, within errors. However, most of the large carbonaceous grains deviate by >2σ in at least one of these isotopic systems (Suppl. Table S1).”\nCollection of additional data would take many additional months, and therefore we hope that the editors accept our changes so as to not delay publication.\n- Concerning the identification of CO3 rich material, it displays a 290.4eV peak assigned to CO3 functional groups, but lacks a C=O peak. In parallel, the authors rule out carbonate minerals as there is a lack of a characteristic, strong, extended fine structure typically observed in A0108-3 (Figure 4). Can you clarify if the latter (due to carbonate minerals) could be also “hidden”, similar to the C=O peak or because of a different process? In particular, such 290.4eV peak has also been observed by Yabuta et al. 2023. Similarly, no fine structure at higher energies (294 to 304 eV) could be observed, and Yabuta et al. concluded that the carbonates were not in a crystalline structure but more likely present as molecular carbonate. They also suggested that the Mg signature observed on clay-bound organics could indicate that Mg “be associated with molecular CO3, MgCO3, or a combination of these”.\nThe reviewer is correctly pointing out the similarity between the CO3-rich organics we describe in our paper and that described by Yabuta et al. (2023) and Stroud et al. (2024). They are the same organic matter. We have added statements to the manuscript that make clearer that we are observing the same material:\n“This abundant molecular carbonate was also previously reported in Ryugu matrix14,41.”\nThe reviewer makes an interesting counterpoint that mineral carbonate EXAFS peaks could be hidden somehow. I can speak from experience that even nanoscale carbonates show clear EXAFS peaks in C-XANES spectra. We have edited the text to include this point:\n“This extended fine structure is present in a calcite grain is observed in the section and within organic matter in the immediate surrounding mineral matrix, presumably taking the form of nanoscale calcite grains.”\nWe have also added extra references to the Yabuta et al. (2023) and Stroud et al. (2024) studies.\n- “and visual TEM confirmation of organic matter in between phyllosilicate sheets (Figure 4D) indicates the presence of an abundant clay-associated polycarbonate material in Ryugu.” => I think Figure 4C should be referenced instead of Figure 4D.\nThis has been corrected.\n- Figure 4C. I would suggest to add some information on the image and the label to clarify what we observe on the image and why it indicates the presence of an abundant clay-associated polycarbonate material.\nWe have added arrows to Figure 4C denoting the locations of interstitial organic matter associated with the phyllosilicate sheets. Accordingly, we have updated the caption as well:\n“(C) Bright-field STEM image of intermixed phyllosilicate sheets and brighter, interstitial organic matter from the region shown in (B). Arrows denote locations of accumulations of CO3-rich organic matter.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a great job in improving the manuscript to make their assignments clearer to the reader, specifically concerning the new \"AA\" type of organic matter. It is also good that they have softened their language accordingly. I still believe that the diffuse type of OM is more widespread in meteoritic samples than the authors claim due to the aforementioned sampling bias, but this discussion is beyond the scope of the paper. I am generally fine with their statement \"..., which is not similarly observed in CI or CM chondrites.\", but maybe they can add something like \"...but more work on this type of diffuse OM is needed in meteorite samples\". I therefore support publication in Nature Communications."}, "author_response": ""}]

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