Continental shelves have the potential to remove atmospheric carbon dioxide via the biological pump, burying it in seafloor sediments. The efficiency of marine carbon sequestration changes rapidly due to variations in biological productivity, organic carbon oxidation, and burial rate. Here we present a high temporal resolution record of marine carbon sequestration changes from a western South Atlantic shelf site sensitive to Brazil Current-driven upwelling. The comparison of biological records to rare earth element (REE) patterns from authigenic oxides shows a strong relationship between higher biological productivity and stronger particle reactive element cycling (i.e. REE cycling) during rapid climate change events. This is the first evidence that authigenic oxides archive past changes in upper ocean REE cycling by the exported organic carbon. In addition, our data suggest that Brazil Current-driven upwelling varies on millennial-scales and in time with continental precipitation anomalies as registered in Brazilian speleothems during the Holocene. This indicates an ocean–atmosphere control on the biological pump, most probably related to South American monsoon system variability. The effectiveness of carbon sequestration in the shallow benthic environment is limited by the biological production, oxidation, and burial rate of organic material2. Despite the achievements in the understanding of the role of recent climate change in carbon sequestration3, it is still poorly known to what extent natural centennial to millennial-scale climate variability of the Holocene (11.7 ka ago to present) impacted carbon sequestration.The production and burial of marine organic carbon play a key role in oceanic biogeochemical processes at shallow and intermediate depths. Primary producers in the surface ocean (i.e. phytoplankton) uptake carbon dioxide that ultimately comes from the atmosphere to produce organic carbon and support the column water food web. The proportion of soft tissue that escapes degradation in the water column and is buried has been termed “marine carbon sequestration”5, as well as its ecological impacts over the deep-sea biodiversity6. The cause of these changes however remain unknown, but changes in solar activity, oceanic and atmospheric heat transport have been proposed7. The interhemispheric pattern of rapid centennial to millennial-scale variability during the Holocene seems to exhibit a “seesaw” behavior, as expressed in the North and South Atlantic. During rapid cold conditions in the North Atlantic, western South Atlantic SST exhibit anomalously high temperatures because of the decreased northward oceanic heat transport8. The rapid changes observed in Southern Hemisphere climatic records suggests that the South American Monsoon System (SAMS) and the western South Atlantic surface ocean circulation varied at same timing11. Both SAMS and the South Atlantic surface circulation are sensitively linked to North Atlantic variability by the Intertropical Convergence Zone (ITCZ), which shows a seasonal meridional migration and a latitudinal contraction following the orbital-scale insolation-driven changes12.Rapid climate changes during the Holocene have been recorded globally on centennial to millennial-scale. They are especially prevalent and well-studied in the North Atlantic through records of freshwater influx and sea-surface temperature (SST)13 and drives physical oceanographic changes along the Brazilian margin. A strong BC fosters the upwelling of South Atlantic Central Waters (SACW) in specific zones of the Brazilian margin, bringing nutrients to the surface and stimulating biological primary productivity. This strengthening has been observed in instrumental datasets14, recent records16, and Holocene reconstructions17. However so far there has been no high temporal resolution record focusing in compare centennial to millennial-scale Holocene changes in the biological pump to the rapid Northern Hemisphere climate events.During the austral summer, when ITCZ is southward displaced, intensified wind stress causes the Brazil Current (BC) to strengthen22, confirming the importance of continental shelves as potential REE sink23. Scavenging by organics also opens raises the possibility that carbon and REE cycles are closely related, opening the possibility of using REE as proxies for carbon sequestration in the ocean. Here we investigate Holocene climate forced export of particulate organic carbon using a benthic foraminiferal productivity record and compare this to REE patterns measured on sedimentary foraminifera with authigenic oxides  from a sediment core collected at the continental shelf of the western South Atlantic. The biological and chemical association were applied to assess the potential of authigenic oxides to record the exported organic carbon control over REE cycling during rapid climate change events.Marine carbon sequestration is closely related to the flux of particulate organic carbon through the water column. Particle flux also controls the distribution of particle reactive trace elements, in particular the fractionation of the rare earth elements (REE). Recent works have suggested that particulate organic carbon can cycle particle reactive trace elements such as REE24.The Cabo Frio Upwelling System (CFUS) is located off the southeastern Brazil and is influenced by the oligotrophic western boundary BC Fig. . The BC 25  during the austral summer (strong BC)25 Fig. . The SAC13. The BC strengthening and its displacement far away from the continental shelf break is in agreement with the SST anomalies observed during the Holocene17. Besides, the SST anomalies in the southeastern Brazilian margin have been related to the intensification of the South Atlantic Convergence Zone over Southeastern South America, a component of the SAMS33.Increases in the BC strength are also coincident with increased SST in the western tropical South Atlantict = 2.87; p < 0.01) and Nd  concentrations. Benthic foraminifera with coatings have slightly higher average trace elements concentrations  than planktonic foraminifera with coatings  , indicating that pure foraminiferal calcite was analyzed Fig. E. Howeve−2 kyr−1, including high values centered at 8.4, 5.6, 5.0, 4.1, 2.6, 2.1, and 1.2 ka  shows millennial-scale variability ranging between 3400 to 741,000 ind cm ka Fig. . The Ce/13C and δ18O) of epibenthic foraminifera Cibicides kullenbergi were relatively constant over the Holocene. The δ13C values vary between 0.57 and 1.48‰, with average value of 1.07 ± 0.15‰. The δ18O results vary between − 0.52 and 1.63‰, with an average value of 0.62 ± 0.22‰  of planktonic foraminifera coatings εNd, which exhibited values between –16.69 ± 0.52 and –15.65 ± 0.29  foraminifera and sedimentary (i.e. CF10-01) foraminifera with authigenic oxide coatings we find that REE concentrations increase by up to tenfold in sedimentary planktonic and 14-fold in sedimentary benthic foraminifera relative to sediment trap planktonic foraminifera  from bottom and pore waters into metal oxides, which precipitate under oxic conditions. The tight coupling of Mn and REE concentrations  reductive dissolution of authigenic oxides; (ii) waters mass advection; (iii) terrigenous inputs; and/or (iv) vertical cycling by particle reactive scavenging. Reductive dissolution of authigenic oxides in the water column can be ruled out because there is no strong oxygen minimum zone in the study area41. In addition, sulphur cycling in CFUS suggests oxic conditions of bottom waters and Holocene sediments38, as well as the strong positive Ce anomaly and the consistently low U/Ca values also do.The source of REE to our study site potentially includes42. If HREE/LREE values were responding to water mass changes, then the higher HREE/LREE values (~ 3.94) could indicate a greater proportion of SACW because it would match the REE pattern of subsurface waters (around 400 m depth) from Brazil Basin42 . The modern signature of SACW 43 is similar to the values reconstructed for the last two centuries in the area. While δ13C and δ18O mean values for the recent and sub-recent epibenthic foraminifera Cibicides kullenbergi are around 1.04 ± 0.10‰ and 0.65 ± 0.24‰16 respectively, our Holocene epibenthic mean δ13C (1.07 ± 0.15‰) and δ18O (0.62 ± 0.22‰) are virtually the same. Although we cannot discard additional influences over the C. kullenbergi δ13C signature , the constant long-term trends of epibenthic foraminiferal δ13C and δ18O suggest that modern bottom water circulation regime should have persisted throughout the Holocene  and present a negligible correlation to the Ce/Ce* . This suggests that the Ce/Ce* and HREE/LREE variabilities were likely caused by a different process other than changes in terrestrial input.Considering the shallow depth and near-shore location of core CF10-01, it is possible that the source of REE could be riverine input. Changes in terrestrial inputs could have changed the MREE/MREE*, which its increment under oxic conditions is often related to input from terrestrial REE sourcesr2 = 0.83; p < 0.01 for BFAR vs. Ce/Ce*, and r2 = 0.50; p < 0.05 for BFAR vs. HREE/LREE) 16. Even though the high accumulation of benthic foraminifera does not indicate the length of organic carbon in the sediments, the BFAR is a strong record of the marine carbon sequestration in millennial-scale during the Holocene.The paleoproductivity proxy BFAR is widely used to reconstruct past organic carbon export to the seafloor39. The scavenged Ce might have been released into pore waters during partial oxidation of organic carbon20 under oxic conditions, so that they are directly incorporated into authigenic oxide coatings. This is an evidence of the potential of the marine organic carbon to scavenge dissolved REE in surface and shallow waters and export them to the greater depths, where they can be released to the seawater impacting its signature.During periods of high BFAR, the REE patterns of foraminiferal authigenic oxide coatings have a higher Ce/Ce* and HREE/LREE values in comparison to the periods of low organic carbon export (i.e. low BFAR) Fig. . The ass37 but instead are reflecting the bottom water mass REE pattern, which is set by vertical export of organic carbon. This also means that, in shallow depths, changes in the Ce/Ce* and HREE/LREE can be used as proxies for marine carbon sequestration by the biological pump. A similar suggestion was made in a GEOTRACES section across the subtropical South Atlantic (at 40oS), where the scavenging dominates desorption at shallow depths (i.e. above 200 m) and the influence of horizontal water mass mixing is only observed in depths below 200 m22. Scavenging removes the LREE from the solution and transfer it to the organic carbon thereby changing the shallow water pattern to high HREE/LREE. Our observation of raised HREE/LREE scavenged pattern and the addition of LREE  recorded by foraminiferal authigenic oxide coatings during periods of high BFAR values suggests its use as a proxy for organic carbon oxidation in the sediment. It also suggests how biological processes can affect Nd cycling in the upper ocean both spatially and though time. The Nd isotope signature of surface and sub-thermocline water masses might be modified by the biological productivity and perhaps suggests a reason why Nd isotopes mimic nutrient tracers in some opportunities47.The REE patterns in foraminiferal authigenic oxide coatings from shallow sediment cores are not integrating the REE scavenged through the water column48. Considering a scenario with lower productivity, we would expect lower Ce/Ce* and HREE/LREE values during the early and mid-Holocene. However, HREE/LREE do not change to the same extent as the Ce/Ce* values during productivity events and very low values are not observed during these periods of lower productivity. These results mean that particulate organic carbon is the preferential phase controlling excess Ce removal from seawater but is not the only phase responsible for the fractionation of the HREE/LREE. The HREE/LREE was fractionated by all sediments  which are always abundant in the location independent of the amount of particulate organic carbon. Therefore, changes in Ce/Ce* are caused by millennial-scale variability of organic carbon which contribute towards but are in addition to background fractionation of HREE/LREE by all particle types.The long-term trends registered in BFAR and Ce/Ce* matches the SST trend in core CF10-01 Fig. C,E,F, su8  are antiphase to SST from CF10-0117  was still transported to our site by longshore drift50, which is then evidenced by our MREE/MREE* and unradiogenic εNd values without the SAMS variability imprint.Our proxy records of changes along the Brazilian margin correspond to precipitation anomalies recorded in speleothem δgth Fig. A. We int10. Considering that the temporal resolution of the BFAR record is not appropriate to track decadal-scale changes but is adequate for millennial-scale changes, we suggest that the millennial-scale climatic variations observed along the Brazilian margin and eastern South America , especially around the ages 4.9, 4.2, 2.7, and 2.0 ka  were analyzed for the last ca. 9 ka 17 was applied here to calculate the BFAR as a product of the sedimentation rate, dry bulk density, and benthic foraminifera density46. REDFIT spectral analysis of the linear detrended BFAR values was performed with the PAST software57 for the last 6 ka, once it is a time-period not affected by the glacial boundary conditions5.To reconstruct past changes in benthic conditions and fauna, the Benthic Foraminifera Accumulation Rate (BFAR) was applied. The sedimentation rate previously determined13C and δ18O) were measured in 10 to 15 specimens of benthic foraminifera Cibicides kullenbergi Parker, 1953 larger than 150 μm. Analyses were performed in a Finning MAT 252 spectrometer at the University of Davis, considering a precision of ± 0.03‰ and ± 0.07‰ for δ13C and δ18O, respectively. Samples were reacted in supersaturated 105% H3PO4 at 90 °C using a Gilson Multicarbon Autosampler. The data were adjusted to Vienna Pee Dee Belemnite (VPDB) using NBS-19 calcite standard. Bracketing stable isotope results correspond to average ± standard deviation.Stable carbon and oxygen isotopes  was calculated as the ratio between Yb and Nd concentrations (ppm/ppm). Cerium (Ce/Ce*) anomaly considered normalized PAAS values60 and was calculated as 3*Ce/(2*La + Nd)61. The middle-REE enrichment (MREE/MREE*) was calculated as [(Gd + Dy)/2]/[(Nd + Yb)/2]62 normalized to PAAS values.Foraminiferal element concentrations were analyzed on 5% aliquots after being dried, re-dissolved in 0.3 M HNONd) in benthic and planktonic foraminifera samples, the REE were separated from other elements using Eichrom TRUspec™ resin, and neodymium was extracted from other REE using Eichrom LNspec™ resin. Samples were analyzed in Neptune Plus multi-collector mass spectrometer (MC-ICP-MS) at the University of Cambridge. Measurements were corrected for mass fractioning to a 146Nd/144Nd ratio of 0.7219. Samples were bracketed with concentration matched solutions of standard JNdi-1 with value of 0.512115 ± 763. Error bars correspond to 2σ of combined internal and external reproducibility of the bracketing standards.For the neodymium isotopes (εSupplementary Figures.


Overall, our results show that the guts of deposit feeders constitute hotspots of organic matter on the abyssal plain that occupy one trophic level above detritus, increasing the food-chain length in this detritus-based ecosystem.Trophic ecology of detrital-based food webs is still poorly understood. Abyssal plains depend entirely on detritus and are among the most understudied ecosystems, with deposit feeders dominating megafaunal communities. We used compound-specific stable isotope ratios of amino acids (CSIA-AA) to estimate the trophic position of three abundant species of deposit feeders collected from the abyssal plain of the Northeast Pacific , and compared it to the trophic position of their gut contents and the surrounding sediments. Our results suggest that detritus forms the base of the food web and gut contents of deposit feeders have a trophic position consistent with primary consumers and are largely composed of a living biomass of heterotrophic prokaryotes. Subsequently, deposit feeders are a trophic level above their gut contents making them secondary consumers of detritus on the abyssal plain. Based on δ Since detritus is the most abundant source of carbon at the base of food webs3, decomposers are the dominant feeding guild on Earth4. Most decomposers are prokaryotes and small invertebrate detritivores5. Thus, detritus-based food webs have highly complex trophic-dynamics and, contrary to primary producer-based food webs, are difficult to compartmentalize into trophic levels. As a consequence, trophic ecology has largely neglected the inclusion of detritus and its pathways into higher trophic levels7, and instead they often appear illustrated as a black box in the flow of carbon or energy through food webs.Detritus is the main standing stock of organic matter for most ecosystems, both aquatic and terrestrial10. Detritus is mainly consumed as it sinks through the water column before reaching the deep seafloor , so these ecosystems are largely limited by food, which often arrives with marked seasonality12. The deep ocean is the largest ecosystem on Earth, and within it the abyssal seafloor occurring between 3000 and 6000 m depth, covers 54% of the Earth’s surface13. Abyssal plains can host high faunal diversity and given their vast size are a major reservoir of biodiversity9, which can exert a significant influence on the carbon cycle. Thus, understanding the energy flow within the deep-sea detrital food web and the trophic ecology of abyssal species is of global importance.Most deep-sea ecosystems, where there is no in situ primary production , entirely depend on detritus arriving from surface waters15. Deposit feeders play a key role in abyssal ecosystems since they rapidly consume detritus reaching the seafloor and are major sediment bioturbators18. They employ an array of feeding and digestive strategies that give them a competitive advantage to cope with food scarcity20. Some species of mobile deposit feeders selectively feed on freshly deposited patches of organic matter22. In other species the gut anatomy allows for efficient processing of ingested food by gut microbes23. In this sense, the guts of some deep-sea holothurians have been found to hold high abundance of prokaryotes relative to the surrounding sediments25. These microbial communities seem to increase the efficiency of deposit feeders to exploit the limiting resources, however, their functional role and whether they are selected from the environment or have a symbiotic origin remain unknown. It has been suggested that these microbial communities act as “commensal” flora to the deposit feeder25, possibly transforming refractory organic matter into compounds easier to digest26, or they might be used as a nutritional source for the deposit feeder27. Overall, gut microbial communities might be a crucial yet overlooked component of abyssal food webs.Deposit feeders are adapted to live in such food-poor environments and unlike many ecosystems, on the abyssal plains they can be very large, dominating megafaunal communities29. The calculation of trophic position using CSIA-AA is based on the differential enrichment in 15N of amino acids (AAs), which can be grouped into “source” and “trophic” AAs30. “Trophic” AAs, like glutamic acid, increase their δ15N values with trophic level, whereas “source” AAs, like phenylalanine, reflect the δ15N values at the base of the food web, changing little with trophic transfer32. The difference in δ15N values between glutamic acid and phenylalanine of an organism is used to estimate its trophic position. Thus, by applying CSIA-AA, information about trophic position and the δ15N values at the base of the food web can be obtained from a single sample. This technique has also been used to prove that in laboratory-cultured heterotrophic bacteria, metabolic reactions can result in changes of δ15N values in AAs similar to metazoans34. Moreover, δ15N values of “source” AAs and δ13C values of essential AAs provide information on dietary sources in consumers. CSIA-AA can therefore be used to estimate trophic position of decomposers and can help to disentangle trophic dynamics of detritus-based food webs.In recent years, compound-specific stable isotope analysis of amino acids (CSIA-AA) has proven useful in estimating trophic position of metazoans more accurately than bulk isotope analysisIn this study we investigated the trophic ecology of abyssal deposit feeders and the role of microbial assemblages present in their guts. We suggest that gut microbes play an important role in supplying nutrition to deposit feeders. For that, we estimated the trophic position of three species of deposit feeders collected from the abyssal plain of the Northeast Pacific , their gut contents and the surrounding sediments using the nitrogen isotopic composition of amino acids. Furthermore, we investigated the role of gut microbes in deposit feeder nutrition using the carbon isotopic composition of essential amino acids. Our findings contribute to the understanding of the trophic dynamics of this deep-sea ecosystem and reveal that microbes present in the guts of deposit feeders might support a detritus-based food web that is longer than expected.S.globosa, E.rostrata, and O.mutabilis, respectively. Thus, deposit feeders were secondary consumers of detritus present in the surrounding sediments. Both foregut and hindgut contents were approximately one trophic level higher than sediments , calculated from the AAs Glx and Phe, was 1.0 ± 0.2 (mean ± SD) for sediments. This confirms that a β value of 3.4‰ used in the calculation of trophic position was appropriate see “”. TP of 15N values of body tissue were also higher than those of sediments for the three species ; however, bulk δ15N values of gut contents were not different from those of sediments . Similarly to δ15N values, bulk δ13C values were comparable between sediments and gut contents but were higher for body tissue . Trophic position was not significantly different between seasons for any species  although O.mutabilis had a mean TP of 3.3 ± 0.0 and 2.8 ± 0.4 in periods of low and high food supply, respectively. However, bulk δ15N values of gut contents of all individuals collected were lower in the more productive period for both O.mutabilis  and E.rostrata .Bulk δO.mutabilis it was three and four times higher in periods of low and high food supply, respectively  to surface sediments was 0.22 weight (wt.) % for both periods. The contribution of TN in the hindgut contents of DFs was twice as high, ranging from 0.32 wt.% to 0.51 wt.%. In the foregut of 13C values of essential AAs (δ13CEAA) showed that PC1 and PC2 together explained 68.3% of the variation . Phe and Val differed significantly between gut contents and body tissue, whereas Lys differed between sediments and gut contents . After including gut contents and sediments as two food sources in the training set, the LDA grouped all DFs collected during low food supply with gut contents (probability > 99%), whereas during high food supply E.rostrata was grouped with sediments (prob. > 90%) and O.mutabilis showed a mixture of both sources (prob. 30–55% of gut contents).Results of PCA of normalized δ15N values of “source” AAs (δ15NSrc-AA) in gut contents and body tissue in O.mutabilis  and S.globosa (11.1 ± 1.9‰) were similar to surrounding sediments (11.6 ± 1.3‰). However, E.rostrata body tissue (15.0 ± 0.3‰) and gut contents in the period of low food supply (18.1 ± 0.5‰) showed higher δ15NSrc-AA values relative to sediments  and S.globosa (0.43 ± 0.42) than in those of E.rostrata  and S.globosa (13.3 ± 3.3%) also indicated a lower degree of degradation than in those of E.rostrata  than in that of high food supply (− 0.94). ΣV values ranged between 2.6 and 4.9, indicating a high degree of heterotrophic microbial resynthesis. However, ΣV values were not significantly different among species or between species and sediments  in gut contents of 15N values in DFs relative to sediments, attributing such values to feeding selection38, to feeding on older, more recycled material35 or to a different trophic 15N-discrimination of ingested microbial biomass37. However, our results based on CSIA-AA, which are not dependent on assumptions of the δ15N value at the base of the food web, indicate that they are secondary consumers. In fact, this was true for the 3 species analyzed, despite their bulk δ15N values differing by 3.5‰, which typically corresponds to a difference of one trophic level39. Abyssal DFs are, therefore, trophically equivalent to zooplanktivorous fish or insectivorous birds.Here we used CSIA-AA to show that megafaunal deposit feeders (DF) were secondary consumers of detritus on the abyssal plain at Station M. This was shown by their trophic position, which was two trophic levels higher than the surrounding sediments. To this date, these are the first estimates of trophic position reported for deep-sea DFs using CSIA-AA approaches. These estimates were higher than expected for organisms feeding mostly on detritus, which are assumed to have trophic positions consistent with primary consumers. Previous research using stable isotope analysis of bulk material has reported high δ17, presence of phytopigments like chlorophyll a in the guts42, higher concentrations of organic carbon and nitrogen in their guts compared to surrounding sediments18, or excess 234Th activities in gut contents similar to sediment traps but higher than surface sediments43. In our study, the gut contents were 18 to 35 times enriched in N from AAs relative to sediments , δ15N and δ13C values of bulk gut contents were not different from those of sediments (but see below for differences among species). If the material present in the guts had been selected and ingested from the environment, their high TP relative to sediments would have been reflected in higher bulk δ15N values as well4. However, 15N was not gained nor lost in the gut. This unchanged isotopic signal of bulk material can only be explained if the increase in TP of gut contents took place after the particles had been ingested. In other words, our results suggest that the estimated TP in the guts corresponded mainly to a living microbial biomass, whereas the bulk δ15N values remained unchanged because they integrated the living biomass with waste products, which became depleted in 15N but do not include AAs.Consequently, the main food source of DF must have a trophic position consistent with primary consumers. This agrees with the estimated trophic position of gut contents, which was around one trophic level higher than that of surface sediments Fig. . One pos44 and guts26 of abyssal holothurians has been previously reported, with abundances 1.5-fold to fivefold higher than the surrounding sediments24. Also, a high RNA:DNA ratio found in the foregut of Oneirophantamutabilis40 supported the idea of a high proliferation of bacteria in their guts. Although this microbial community could be selected from the environment, our hypothesis that they primarily grow inside the gut was supported by δ13C values of essential AAs (δ13CEAA). We found that surface sediments and gut contents had distinct δ13CEAA patterns  were associated uniquely with the guts. Overall, guts of mobile megabenthos concentrate large quantities of organic matter that are largely the living biomass of heterotrophic prokaryotes that form a unique community in the guts.A remarkable abundance of bacteria in tentacles26, where most absorption of nutrients occurs46. The presence of a flourishing microbial community in hindguts of DFs, later released as fecal pellets to the environment, might create microbial hot spots and contribute to the microbial productivity on the abyssal plain.It is noteworthy that the TP of foregut contents was similar to that of hindguts Fig. . Moreove49. However, using counts of bacteria in the guts, the calculated prokaryotic production and the gut transit time, it has been estimated that, overall, prokaryotic biomass contributes only between 0.1–3% of the total protein taken up by deep-sea holothurians27. In our study the trophic position estimated for the studied DFs, one trophic level above their gut contents, led us to hypothesize that DFs were assimilating AAs from the prokaryotic biomass inhabiting the guts. However, δ13C values of essential AAs in the body tissue of DFs, which come necessarily from the diet, were a combination of those of sediments and gut contents, pointing also to the assimilation of AAs directly from ingested detritus . Echinocrepisrostrata also carves trails at a depth range of 0–2 cm54, so it is likely that they ingest sediment below 0.5 cm. However, this was not reflected in their isotopic composition because sediments showed a similar isotopic composition downcore  than gut contents of E.rostrata, supporting the selection of fresher organic matter by the two holothurians.We also found differences between the three species analyzed, despite the general patterns described. The elasipod holothurians V parameter, which is an index for microbial heterotrophic resynthesis56, was comparable between gut contents of the three species and sediments . The seasonality of food supply has an effect on the abundance59 and reproduction54 of megafauna, but the overall trophic identity of the studied DFs as secondary consumers was maintained. However, we observed some differences in the isotopic composition of gut contents. Gut contents of both O.mutabilis and E.rostrata showed higher bulk δ15N values during the period of low food supply (i.e. May). For O.mutabilis that difference appeared to be trophic  were slightly more degraded  they were more similar to those of sediments in E.rostrata and a mixture of both in O.mutabilis , such differences were not reflected in the elemental or stable isotopic composition of surface sediments. In fact, the DI, the Σed Table . It is llis Fig. B. Previo9. In this ecological framework, the role of prokaryotes as primary consumers might be crucial in providing stability to the food web. As a result, gut microbes deserve attention in future studies analyzing energy flows in the food web of abyssal plains.Our study revealed for the first time that DFs inhabiting the abyssal plain are secondary consumers of detritus, in contrast to the common assumption that their nutrition comes directly from detritus. The gut contents of the three species analyzed are trophically distinct from the surrounding surface sediment and their mix of prokaryotic biomass and detritus form a unique food source, which is one trophic level higher than sediments. Our results suggest that gut contents are mainly composed of a heterotrophic microbial community, which is assimilated as a food source by the DFs most importantly in periods of low food supply. In summary, we found that microbial communities in the guts of deposit feeders play a key role as primary consumers in the food web of the abyssal plain. The trophic dynamics in this deep-sea ecosystem are bottom-up controlled by the flux of organic matterAlvin and the ROV DocRicketts, respectively. Upon retrieval to the surface, samples were placed in a cool room (5 °C) for their further processing.Samples were collected from the abyssal plain (~ 4000 m) in the Eastern North Pacific off of the California coast . Sediment cores and megafauna were collected in May and October 2019 using the HOV Oneirophantamutabilis complex, Scotoplanesglobosa and echinoid Echinocrepisrostrata were weighed and measured, then they were dissected using an scalpel. We chose for this study three of the most abundant species of deposit feeders at Station M 59 with a large size so that we could sample the amount of gut contents required for the analyses (~ 150 mg in dry weight). We made a longitudinal cut along the digestive tract and took a sample of the foregut and hindgut contents, avoiding gut tissue. Then we removed the remaining guts and took a sample from cleaned body tissue, or in the case of echinoids, from the test. All samples were placed in cryovials and frozen in liquid nitrogen, and subsequently stored at − 80 °C.Sediment cores of 7 cm diameter were sliced in depth intervals , placed in petri dishes and stored frozen at − 80 °C. Specimens of holothurians 13C and δ15N values verified independently in other laboratories) were typically of the order of less than ± 0.2‰ for δ15N and δ13C values. In elemental analyses, the variation of repeated measures performed in a subset of samples was < 10%.Samples of sediments, gut contents and body tissue were freeze dried and ground to a homogenous powder using mortar and pestle. For analysis of bulk nitrogen and carbon isotopic composition, TN and TOC, ~ 0.7 and 3 mg of body tissue from holothurians and echinoids, respectively, ~ 5 mg of gut content and ~ 20 mg of sediments were placed in silver capsules. Samples were acidified to remove carbonates with 1 M HCl, which was added dropwise until bubbling ceased, then dried at 60 ºC and packed. Nitrogen and carbon elemental composition and isotopic composition were determined simultaneously using an isotope ratio mass spectrometer (DeltaPlusXP or Delta-V-Advantage) coupled to an elemental analyzer (Costech Model 4010). Measurement error based on within-run replicates of reference materials . Samples were analyzed following the methods of Hannides et al.60. Briefly, samples were hydrolyzed using trace metal-grade 6 N HCl and then purified using cation exchange chromatography. Samples were then esterified using 4:1 isopropanol:acetyl chloride and derivatized using 3:1 methylene chloride:trifluoroacetyl anhydride. To account for carbon added during derivatization and variability of isotope fractionation during analysis, we also derivatized and analyzed a sample containing a set of 14 pure AAs  purchased commercially . The resulting trifluoroacetyl and isopropyl ester derivatives were purified using chloroform extraction and stored at − 20 °C until analysis.For δ13C values were corrected based on the analysis of the set of pure AAs prepared under the same conditions following the approach by Silfer et al.63. Nitrogen isotopic composition of AAs was analyzed using a Thermo Scientific Delta V Plus IRMS interfaced to a trace gas chromatograph (GC) fitted with a 60 m BPx5 capillary column through a GC-C III combustion furnace (980 °C), reduction furnace (680 °C) and liquid nitrogen cold trap.Carbon isotope composition was measured using a Thermo-Fisher Scientific MAT 253 isotope ratio mass spectrometer interfaced with a Trace Ultra GC-III via ConFlo IV. δ15N and δ13C values, was typically < 1‰. For replicate injections, δ15N and δ13C standard deviations averaged 0.4‰ and 0.5‰, respectively. Results for each sample are given as the mean values of all injections. We obtained information for the following AAs: alanine (Ala), aspartic acid , glycine (Gly), glutamic acid , isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), and valine . Full AA reference suites of known weight were injected every 3 sample injections. The corresponding response factors (Vs [nmol AA]− 1) were used to determine total AA concentration and the contribution of each AA to the total AA pool . δ15N values were normalized using the fitted regression line between the known δ15NAA values and the measured δ15NAA values of the AA reference suite injected prior and after every 3 sample injections.When possible, each sample was measured on 3 replicate injections (but sediments were only injected once due to their low AA content), with internal reference materials norleucine and aminoadipic acid with known isotopic composition co-injected on each run. The accuracy of CSIA-AA measurements, as the difference between the isotopic composition of the internal reference materials co-injected on each run and their known δ15N values of “source” AAs (δ15NSrc-AA) were calculated as the average δ15N value of: serine, phenylalanine, lysine and glycine. Trophic position (TP) was calculated using the equation from Chikaraishi et al.31: TP = (δ15Nglx – δ15Nphe – 3.4)/7.6 + 1, where δ15Nglx and δ15Nphe are the δ15N values of glutamic acid and phenylalanine, respectively, 3.4 ± 1.0‰ is the difference between glutamic acid and phenylalanine in primary producers (β), and 7.6 ± 1.2‰ is the trophic discrimination factor (TDFAA). Uncertainty in the calculation of δ15NSrc-AA and TP due to analytical error and uncertainties in TDF and β values was calculated by propagation of errors . To determine whether sediments and gut contents constituted distinct food sources we focused on δ13C values of essential AAs . We normalized the δ13C values of each essential AA to the mean value of all essential AAs for each sample. Algae and bacteria have highly conserved modes of amino acid biosynthesis that produce unique patterns of carbon isotope fractionation. This allows the origins of essential amino acids to be determined from a comparison of the distribution patterns of δ13C values between samples since they fall on the same scale around an average equal to zero45.δV parameter is a proxy for heterotrophic resynthesis of AAs and is defined as the average deviation of δ15N values of individual trophic AAs from the mean δ15N value of those AAs56. The ΣV was calculated as: ΣV = 1/n Σ Abs (χi), where n is the total number of AAs used for the calculation and χi is the deviation of the δ15N value of amino acid i from the mean δ15N value of the n amino acids [ δ15Ni – (Σ δ15Ni /n)]. We used five AAs to calculate ΣV , excluding Ile because its δ15N value was missing in a few samples. Nevertheless, for those samples with estimated δ15N values of the six AAs the ΣV parameter was not significantly different when estimated with and without Ile since the slope of the ΣV6-AAvs. ΣV5-AA relationship  was not significantly different from 1 [slope = 0.98 ].The Σ65. In this sense, Gly is abundant in cell walls so its high molar abundance (Mol% Gly) also indicates higher degradation state of particles or sediments66. The DI was calculated following the formula proposed by Dauwe et al.65: DI = Σ [(vari – AVG vari )/STD vari] × fac.coefi, where vari is the Mol% of amino acid i in our dataset, AVG vari and STD vari are its mean and standard deviation in the reference dataset (based on a variety of samples from marine phytoplankton to deep-sea sediments) and fac.coefi the factor coefficient from amino acid i based on the first axis of the PCA. AVG vari, STD vari and fac.coefi were obtained from Table 1 in Dauwe et al.65.The degradation index (DI) is based on the selective preservation of AAs, so that AAs that comprise refractory material like cell walls will be more abundant in degraded material15N values between types of samples . To disentangle the differences in patterns of δ13C values of EAAs  we used multivariate statistics. We performed a principal component analysis (PCA) to the normalized δ13C values of each AA, then we used the permutational analysis of variance (PERMANOVA) to find statistically significant differences in δ13C values of EAAs between groups. We also performed a Linear Discriminant Analysis (LDA) using δ13CEAA values of sediments and gut contents as a training set to determine whether δ13CEAA values of body tissue grouped more closely with sediments or gut contents. All analyses were performed using R version 3.6.367.We used analysis of variance (ANOVA) to study differences in TP increment or bulk δSupplementary Figures.


During the electrochemical lithiation and sodiation, the formation of Y5-xPrxSb3-yMyLiz and Y5−xPrxSb3−yMyNaz maximally disordered–high entropy intermetallic phases (MD-HEIP), as the result of insertion of Li/Na into octahedral voids, were observed. Carbon nanotubes (CNT) are an effective additive to improve the cycle stability of the Y5−xPrxSb3−yMy  anodes for lithium-ion (LIBs) and sodium-ion batteries (SIBs). Modification of Y5−xPrxSb3−ySny alloys by carbon nanotubes allowed us to significantly increase the discharge capacity of both types of batteries, which reaches 280 mAh · g−1 (for LIBs) and 160 mAh · g−1 (for SIBs), respectively. For Y5−xPrxSb3−yPby alloys in which antimony is replaced by lead, these capacities are slightly smaller and are 270 mAh · g−1 (for LIBs) and 155 mAh · g−1 (for SIBs), respectively. Results show that structure disordering and CNT additives could increase the electrode capacities up to 30% for LIBs and up to 25% for SIBs.The maximally disordered (MD) phases with the general formula Y During the electrochemical lithiation and sodiation, the formation of Y5−xPrxSb3−yMyLiz and Y5−xPrxSb3−yMyNaz maximally disordered–high entropy intermetallic phases, as the result of the insertion of Li/Na into octahedral voids, were observed. Carbon nanotubes are an effective additive to improve the cycle stability of the Y5−xPrxSb3−yMy  anodes for lithium-ion and sodium-ion batteries. Modification of Y5−xPrxSb3−yMy  alloys by structure disordering and carbon nanotube additives allowed us to significantly increase the discharge capacity of both types of batteries, which reached 280 mAh · g−1 (for LIBs) and 160 mAh · g−1 (for SIBs), respectively. A CNT-modified electrode also shows higher corrosion resistance in electrolyte solutions and, as a result, demonstrates an increased battery life. The effect of the addition of CNTs and PVDF binder on the improvement of capacity and cycle stability of electrodes is due to the following factors: CNTs provide diffusion pathways for Li+ or Na+ ions to most of the grains inside the material, improving charge/discharge kinetics; PVDF provides good adhesion between the electrode materials and current collectors, giving excellent electrochemical, mechanical, and thermal stability of electrodes.The maximally disordered Y


Carbon isotopic analysis is among the most pervasive geochemical approaches because the fractionation of carbon isotopes produces a natural tracer of biological and chemical processes. Rover-based carbon isotopic analyses of sedimentary rocks on Mars have the potential to reveal modes of Martian carbon cycling. We report carbon isotopic values of the methane released during pyrolysis of samples obtained at Gale crater. The values show remarkable variation indicating different origins for the carbon evolved from different samples. Samples from multiple locations within Gale crater evolved methane with highly fractionated carbon isotopes. We suggest three routes by which highly fractionated carbon could be deposited on Mars, with each suggesting that Martian carbon cycling is quite distinct from that of the present Earth. 13CVPDB) of the methane released during pyrolysis of 24 powder samples at Gale crater, Mars, show a high degree of variation (−137 ± 8‰ to +22 ± 10‰) when measured by the tunable laser spectrometer portion of the Sample Analysis at Mars instrument suite during evolved gas analysis. Included in these data are 10 measured δ13C values less than −70‰ found for six different sampling locations, all potentially associated with a possible paleosurface. There are multiple plausible explanations for the anomalously depleted 13C observed in evolved methane, but no single explanation can be accepted without further research. Three possible explanations are the photolysis of biological methane released from the subsurface, photoreduction of atmospheric CO2, and deposition of cosmic dust during passage through a galactic molecular cloud. All three of these scenarios are unconventional, unlike processes common on Earth.Obtaining carbon isotopic information for organic carbon from Martian sediments has long been a goal of planetary science, as it has the potential to elucidate the origin of such carbon and aspects of Martian carbon cycling. Carbon isotopic values (δ Curiosity rover of the fluvio-lacustrine sedimentary system at Gale crater provides unique opportunities, as samples are collected from a variety of locations within a known stratigraphic context   context , 2. MSL  context  from 24 −1 under a helium flow in quartz cups up to ∼850 °C. A fraction of the evolved gas mixture is directly analyzed with a quadrupole mass spectrometer, and a specific preselected temperature cut of the remaining gas is diverted to the Herriott cell of the TLS. The redox state of the oven, in practice, depends on the relative abundances of oxychlorine species and reduced minerals in each sample. During EGA, organic material can produce a range of products including CO2, CH4, CO, OCS, CS2, and molecular organic fragments. EGA δ13C CH4 values were measured by the TLS instrument of the SAM suite for 24 samples from Gale crater, Mars .For evolved gas analysis (EGA) at Gale crater, drilled powder or scooped fines are heated at 35 °C miner, Mars  using me4 δ13C values that are highly 13C depleted correspond predominantly with the 34S-depleted δ34S QMS values observed for evolved SO2, reported by Franz et al.  temperature has been interpreted to indicate Martian sulfides  in evolved CH4 also evolved sulfur gases (OCS and CS2) indicative of a reduced sulfur detection based on statistical comparisons to laboratory runs  are exceptionally clean and provide multiple 12C and 13C lines with which to calculate δ13C values, making it unlikely that the 13C depletions observed are due to an interfering organic molecule. From the repeat CB analyses, it appears that the isotope depletion observed in CH4 is most pronounced at lower temperatures. This observation suggests that precursors to the evolved CH4 are relatively volatile organic molecules. However, strong depletions were still observed in several samples using high-temperature cuts (>450 °C) in which the temperature cut includes a tail of a CH4 release centered at a lower temperature . The CH4 isotopic variation in CB samples, though, are not completely explained by differences in the temperature cut. Additionally, at Yellowknife Bay, the TLS analyses of CB using the 2.78-μm laser produced highly 13C-depleted CO2 δ13C values . While these TLS analyses of CB produced CO2 δ13C values that were, in some cases, comparable to CH4 δ13C , such depleted CO2 δ13C values have not been observed in later samples of the mission. In contrast, TLS CH4 δ13C values using the 3.27 μm laser show strong depletions in multiple different drill samples from vastly different parts of the mission. Because the observation of anomalous 13C values in evolved CH4 are repeatable with different samples spread out in space and time, we have focused on those values for this study.Some of the is ∼+46‰ . This atN-tert-butyldimethylsilyl-N-methyl-trifluoroacetamide -1,1,3,3-tetramethyldisiloxane [or bisilylated water (BSW)], which can be monitored by the SAM QMS. Based on the levels of BSW detected, the level of MTBSTFA background has been variable between runs depending on how long it has been since a wet chemistry experiment and, for a few cases in which it was relevant, how long a sample was stored before analysis. For 16 of the samples, the mean of the total BSW observed during the course of the EGA runs was 1.0 nmol with an SD of 1.5 nmol  similar to MTBSTFA (δ13C = −35‰), which demonstrated that with elevated levels of MTBSTFA in the SAM background, a portion can end up as evolved CH4. Further, an intramolecular isotopic analysis of methyl-trifluoroacetamide from the hydrolysis of MTBSTFA showed that the types of carbon (methyl-carbon and carbonyl C) most likely to contribute CH4 from the MTBSTFA background were the most 13C enriched , making it unlikely that the observed anomalies stemmed from a site-specific carbon depletion obscured in the bulk δ13C value for MTBSTFA.We have considered the SAM instrument background of yrolysis . In addi1.5 nmol . HU had SI Appendix, Table S4) presently on Mars are the atmospheric CO2 . We found that cleavage and reduction of methyl groups to CH4, as would happen for most CH4 derived from MTBSTFA products, produced little 13C depletion . CH4 evolved from recalcitrant sources (graphite and diamond) showed 13C-depletion of 8 to 21‰, CH4 from oxalate/oxamide showed moderate 13C depletion (25.0 to 25.3‰), and bicarbonate reduction showed the largest 13C depletions (28.5 to 49‰). Both our laboratory pyrolysis experiments  and modeling of possible isotopic pseudoequilibration during pyrolysis  considering several different scenarios suggested that oven processes would typically produce fractionations less than 50‰ and, therefore, cannot account for the large 13C depletions observed in multiple samples at Gale crater. Applying the most extreme oven fractionation imagined to these carbon reservoirs results in CH4 with δ13C values of about −5 to −70‰. CB , Gobabeb (GB2), Highfield (HF), Rock Hall (RH), Hutton (HU), and EB showed TLS CH4 values more 13C depleted than this range, indicating that oven reactions are not likely to be causing their anomalous 13C-depleted values observed in evolved CH4.Major endmember carbon reservoirs , a modern dune field of basaltic sand  found for 10 of the Gale crater samples.One potential connection between these evolved methane samples depleted in osurface  and 4. Tnformity . Similar13C-depleted signature may be a coincidence, it could be explained if the signature relates to a process that primarily impacted a paleosurface associated with outflow from Gediz Vallis and Peace Vallis that included the Greenheugh pediment capping unit, the topographic high represented by the VRR, and the crater floor at Yellowknife Bay. Given this distribution of samples yielding isotopically anomalous methane, we have considered multiple phenomena that have the potential to explain the results. The phenomena discussed are not presented in order of preference, but rather are treated equally, starting with the canonical explanation for when this type of geochemical signature is found on Earth and moving to possible processes that would be increasingly specific to Mars.While this distribution of samples yielding a strongly 13C-depleted EGA CH4 was presumably released from organic materials in the solid sample during pyrolysis. The canonical explanation on Earth for highly depleted carbon associated with a paleosurface would be microbial methanotrophy converting methane into biomass when the methane was already 13C depleted because of its biological production during methanogenesis  because the observed depletion on Earth during anaerobic methanotrophy is on the order of about 30‰  once every 100 million years, providing a mechanism for triggering cooling events on terrestrial planets through the influx of particles to planetary atmospheres that is substantially higher (20 to 100 times) than the ongoing flux of interplanetary dust . The solarticles , as abouarticles . The δ13as −260‰ , demonstchannels –44.13C-depleted values at Gale crater largely fits the observations if the isotopic signatures are not diluted by other geological processes and with the understanding that this explanation relies on a rare event, making it perhaps coincidental that the mission should find this signature preserved. Finally, the SAM results presented here suggest that the most 13C-depleted values are liberated during relatively low oven temperatures, with the high-temperature examples typically being tails of lower-temperature methane release. These observations might be understood to indicate that the organic precursor molecules being converted into methane in the SAM oven are not recalcitrant kerogen-like molecules, as pyrolysis of kerogen usually occurs at a higher temperature. If this interpretation is correct, this constraint can be consistent with an interstellar dust origin, as there are a wide variety of known interstellar molecules, including many that are highly volatile , and so either the abiotic reduction would need to have been occurring in subsurface environments (which does not match our observations) or would need to have occurred prior to the loss of much of the Martian atmosphere . While the timing of the erosional paleosurface is poorly constrained to the Hesperian, deuterium enrichment of clays at Gale crater indicated significant loss of atmospheric volatiles by this time . Fractionation of this magnitude would not produce organic material as depleted as the observed TLS values for the EGA methane reported in 13C-depleted evolved methane during pyrolysis of samples at Gale crater. However, carbon isotopic fractionation during photochemical reduction of CO2 on a mineral surface has not been experimentally evaluated, and so, further research is needed before we can fully evaluate the abiotic reduction as the source of the highly 13C-depleted carbon isotopic values reported here. To be clear, there are a number of other CH4 isotope values reported in 2 on the Martian surface, including the most 13C-enriched values (−1 ± 6‰ to +22 ± 10‰). These 13C-enriched results match what would be expected for processes, biotic or abiotic, that produce organic material from the modern Martian atmosphere, for example.There is growing recognition of the possible abiotic production of organic carbon on Mars through either electrochemical reduction  or photohis time , and thehis time . Overall13C depletions observed by the MSL-TLS component of the SAM instrument.Photochemical reactions are attractive as a source for the observed phenomenon because photochemical reactions are known to have influenced Mars’s volatiles, including the sulfur isotopic values found in Martian meteorites –52. Also4 abundances in the Martian atmosphere  representative of carbon from the interior of Mars. In order to produce organic material that is highly 13C depleted (considerably more depleted than −70‰), another step with significant δ13C fractionation would need to have occurred during the formation of the methane. In principle, this could be abiotic CO2 reduction in the Martian crust during serpentinization or microbial methanogenesis. As noted previously, the abiotic reduction of CO2 results in ∼50‰ 13C depletion. If the source CO2 had a δ13C composition of −20‰, subsequent abiotic reduction to CH4, and subsequent photochemistry, then the resultant organic material deposited could be as depleted as perhaps −85‰, a value that does not adequately include all of the values observed at Gale crater. If microbial methanogenesis were producing the observed atmospheric CH4, photolysis of the biologically produced CH4 would result in isotopic compositions consistent with the strongly depleted 13C values observed. At a minimum, however, new observations would be needed to confirm a biological origin for Mars CH4 plumes before this explanation can be accepted.A growing number of studies have reported variable CHmosphere –55, inclmosphere , 57 and an Earth . Further, Halevy  suggeste2 to formaldehyde (CH2O) via CO as an intermediate  radiation around 100 nm from a synchrotron source . As explained further in the supplement, quadratic discriminant analyses of the QMS gas results provided posterior probabilities for whether each drill sample contained reduced sulfur  is flowed through the pyrolysis oven. After evacuation of the TLS sample (Herriott) cell to record empty cell values, the TLS cell is opened to receive a “temperature cut” ingest  during EGA that introduces He and evolved volatiles into the cell, which is then sealed off for analysis. Typically, empty-cell methane abundances are extremely low (parts per billion by volume) compared to the full-cell EGA values (tens of parts per million by volume [ppmv]) so that empty-cell corrections are not needed. Full-cell pressures (mainly helium) are typically 5 to 15 mbar.12CH4 lines and four strong 13CH4 CH4 lines are identified in the recorded TLS spectra, although only two of the 13CH4 lines are chosen for analysis to minimize water interferences . With the cell closed, the laser scans over the methane lines every second, and on-board, TLS captures average spectra over sequential 2.7-min periods that are downloaded. Analysis of each of these 2.7-min spectra is done on a line-by-line basis through comparison with HITRAN 2016 calculations  in support of this paper, including an NMR-based investigation (4 under conditions similar to that of the SAM instrument. Various carbon-containing materials were placed in silver boats and dropped into an oven at 400 °C under flowing helium. The oven temperature then increased to >850 °C, and evolved gases, including CH4, were trapped out of the He flow between 455 °C and 755 °C to simulate a high-temperature TLS cut.As discussed in the tigation  of the iSupplementary File


Functional particles that respond to external stimuli are spurring technological evolution across various disciplines. While large-scale production of functional particles is needed for their use in real-life applications, precise control over particle shapes and directional properties has remained elusive for high-throughput processes. We developed a high-throughput emulsion-based process that exploits rapid vitrification of a thixotropic medium to manufacture diverse functional particles in large quantities. The vitrified medium renders stationary emulsion droplets that preserve their shape and size during solidification, and energetic fields can be applied to build programmed anisotropy into the particles. We showcase mass-production of several functional particles, including low-melting point metallic particles, self-propelling Janus particles, and unidirectionally-magnetized robotic particles, via this static-state particle fabrication process. Upscale fabrication of functionalized microparticles is a pending challenge. Here, Kim et al. exploit the rheology of a thixotropic medium to grind sizeable amounts of raw material into well-defined colloidal dispersions, physically stabilized for further production steps. Such minute fragments of matter are often incorporated into bulk materials to achieve multifunctional composites11, dispersion inks13, and granular metamaterials14, which yield gained functionalities responsive to external stimuli. Thus, mass manufacturing of functional particles is becoming increasingly important to translate particulate material discoveries to large-scale applications. One widespread method to mass-produce particles from bulk matter is emulsion processing17 that consists of emulsifying a bulk liquid in a continuous phase via either shear mixing or sonication, then solidifying the distributed phase. Although rapid and scalable, previous emulsion processes have not addressed the need for high process yield18, monodispersity20, or anisotropy22. Similarly, micro-fluidic particle fabrication processes can address these critical demands, but produce small numbers of particles (large quantity production is time-consuming and costly).Particles are extensively used in various disciplines, such as medicine24. This phenomenon is usually prevented by stirring the continuous phase until the distributed droplets are fully solidified27, but this redundant shearing causes the droplets to collide and deform, ultimately leading to low process yield and high particle heterogeneity. One proposed solution to this challenge is to fix and isolate each droplet in place by vitrifying the continuous phase after emulsification. This static-state particle fabrication process can eliminate coalescence and droplet deformation, but existing methods require secondary steps to vitrify the continuous phase  which are too complex and laborious to scale production volume.Throughout an emulsion process, sedimentation or creaming of the distributed phase (emulsion droplets) after their creation and prior to their solidification result in droplet coalescence, causing irregular clumps of particles and potentially ruining the overall particle batchHere, we report a method of fabricating particles via a scalable, static-state emulsion process. The method uses a shear-thinning thixotropic medium as the continuous phase that reduces its viscosity with increasing shear and then rapidly vitrifies when the shearing stops. This rheological response of the continuous phase enables both dynamic-state emulsification (during shear mixing) and static-state solidification (with the mixer disengaged), all within the same batch and without additional processing steps. Moreover, once the particles are held in place in the continuous phase, energetic fields can penetrate the continuous phase to induce anisotropy in the solidifying particles, enabling scalable programming of the intrinsic particle properties. Our method herein is highly generalizable as long as the continuous phase exhibits thixotropy, and in this study, gelatinized corn starch in water is used as the medium, which carries the additional advantage of being eco-friendly with sustainable by-products.ω increases, the viscosity ηc of the continuous phase drops and emulsifies the target material. The shearing is stopped after the emulsification and ηc increases by ~100 times to rapidly vitrify the continuous phase. The emulsion droplets are seized by networks of starch within the vitrified medium and remain suspended without creaming, sedimentation, or colliding with each other during the subsequent solidification , a Bi–In–Sn alloy with a low melting temperature of 62 °C, as the distributed phase, as shown in Fig. e.g. ellipsoid, platelet, spiky sphere), and their sizes have a large standard deviation. We hypothesize that such heterogeneous particle shapes and sizes originate from high energy collisions of molten and solidified FM droplets, which is effectively mitigated when starch is present in the medium. The weakly perturbed environment of our static-state process also alters the microstructure of FM, enabling precise control of the particle quality  decreases ηc. Therefore, to retrieve prepared particles, we utilize hydrolysis of starch by adding enzyme, thereby upholding the eco-friendly and biocompatible process initially granted from using starch.We further investigate the underlying mechanism to optimize the processing parameters which will enable mass manufacturing of complex particles Fig. . We drawe ω Fig. . The incϕ. We use this plateau as the maximum value of the viscoelastic stress because it indicates that the medium remains stationary as long an external stress is less than the plateau value. We define this plateau value as the vitrification stress σv (see Methods for details), and since σv monotonically increases with ϕ, the medium can thus be tuned to support heavier droplets.However, distributed droplets are still prone to sediment within the vitrified medium unless their weight can be supported by the viscoelastic stress of the vitrified medium. To measure the maximum value of the viscoelastic stress, the stress relaxation of the starch/water medium is examined Fig. . The strσv, the density of the emulsion droplets V is the droplet volume, g is the gravitational acceleration, and A is the droplet cross-sectional area. Equation  can be indefinitely suspended within the vitrified medium. Figure dv of FM droplets (3) distributed within the starch/water medium, which reveals the ability of the vitrified medium to hold the heavy metallic droplets in place without sedimentation. For example, dv of the FM droplets is ~200 μm at ϕ = 10 wt%, and only ~ 0.1 μm at ϕ = 5 wt%.The vitrification stress mentclass2pt{minimdv. The droplet diameter can be controlled during the emulsification primarily by tuning either the viscosity of the continuous phase ηC or the shearing rate ω. Figure ϕ under a fixed ω, because higher ϕ leads to higher ηC. Therefore, higher ϕ is generally advantageous for enabling vitrification because it decreases dv , but increasing to ϕ = 12.5% allows for droplet suspension in the vitrified medium (dv = 360 μm). In the case of a fixed ϕ, increasing ω can reduce ϕ and ω can be controlled to satisfy the critical process condition of dv.We examine how to control the diameter of emulsified droplets, such that the mean droplet diameter see Fig. . For exaentclass1pt{minima3), enabling dv to reach tens of millimeters while maintaining compatibility with the vitrification process. For the emulsification, particle size analysis shows that SC particles are significantly smaller than DS particles under identical emulsification conditions .Therefore, the coalescence frequency 41. Our static-state method addresses these challenges of mass-production by exploiting the relative difference in the To showcase the far-reaching impacts of our static-state particle fabrication method, we demonstrate the scalable programming of anisotropy in particles, as shown in Fig. res Fig. . The vit3) arranged on one side of a larger PDMS droplet (3). These Janus particles can be used as self-propelling particles when they are placed in hydrogen peroxide, because the Ag catalyzes a process that generates oxygen bubbles  and energy-efficient (no extra power consumption from redundant stirring), which will streamline the technology transfer of this static-state particle fabrication to industry-scale manufacturing.Corn starch granules (MFG 2006779) were purchased from Argo , and used to prepare starch/water mediums by mixing with DI-water at various concentrations of 5–25 wt% and gelatinizing the granules at 95 °C for 1 h. Amylase enzyme (SKU: 5BHBZZ7100B) was purchased from BSG . Field’s Metal blocks (SKU: LMP144) were purchased from RotoMetals  and used as-is. Two-part silicone resin EcoFlex 00-30 and one-part RTV silicone resin Semicosil 964 were purchased from Smooth-On  and Wacker , respectively. Iron powder (<10 μm) (SKU: 267953) and silver-coated ferrite particles (~25 μm) (SM25P20) were purchased from Sigma Aldrich  and Potters Industries, LLC respectively, and used as fillers of anisotropic particles. Hydrogen peroxide and Triton X-100 (SKU: X100) were purchased from Sigma Aldrich  and used as reaction medium and surfactant for propelling Janus particles.A bulk of 10 g Field’s Metal was added to the gelatinized starch/DI-water medium of 200 ml and melted for 2 min at 95 °C. A shear mixer broke up the molten Field’s Metal at various shear rates and removed off after 10 min. Two types of shear mixer were used to emulsify the molten Field’s Metal: a 10-speed overhead mixer  with a 3-bladed stirrer of 56 mm in diameter , and a 6-speed lab homogenizer  with a 20 mm saw-tooth generator . Applied shear rates are ranging from 600 rpm to 9,500 rpm when using the overhead mixer, and from 17,500 rpm to 24,000 rpm when using the homogenizer. The entire medium containing micro-droplets of the molten Field’s Metal was cooled down at room temperature to solidify the molten metal.An aqueous solution of 5 wt% amylase enzyme of 100 ml was then added to the resulting batch and the temperature was raised to 40 °C to catalyze hydrolysis of the contained gelatinized starch. The hydrolysis reaction was performed for 1–2 h under a gentle shearing  to ensure the contact between the enzyme and the starch. Depolymerization of the starch from the hydrolysis caused Field’s Metal particles to rapidly sediment and the particles were easily retrieved. Such a hydrolysis step was especially important for fabricating smaller/polymeric particles because long-chains of the pristine starch remain adhered to the particles until being cut into the short-chain glucose. Retrieved particles were additionally washed using DI-water for 3 times and dried at room condition.t = 1 h, achieved of the logarithmic experimental data. The surface tension of polymer resins in water was measured via a pendant drop method at room temperature. A drop of the resin formed at an ID 0.34 mm syringe nozzle  was imaged using a Grasshopper camera  and an imaging software ImageJ analyzed the radii of the drop. The same method was applied, but the water at 70 °C, to measure the surface tension of the molten Field’s Metal. Optical micrographs of particles were obtained by Smartzoom 5 from Zeiss and scanning electron micrographs (SEM images) were obtained using SU-70 from Hitachi where energy-dispersive X-ray spectroscopy (EDS) was also performed at 10.0 kV. XRD data were obtained using Rigaku SmartLab X-ray diffractometer equipped with Cu Kα radiation under Bragg-Brentano focusing mode at step 0.02° over a 2θ range of 10° to 100°. The phase identification of the particles was analyzed using PDXL software.Rheological behavior of thixotropic gelatinized starch/DI-water medium was measured using RSA-G2 from TA Instruments with a parallel plates fixture of 15 mm in diameter. Frequency sweep was performed at 80 °C with an oscillatory strain of 1.0%, and a solvent trap was used to prevent the premature evaporation of DI-water during the measurements. Stress relaxation was performed at room temperature with the initial strain of 1.0% and k is the Weibull shape factor and λ is the scale factor.The particle size distribution was achieved by two different methods: sieve analysis and imaging analysis. Sieve analysis was performed by sieving a batch of fabricated particles using a set of sieves and constructing a relevant cumulative distribution curve. The mesh sizes of used sieves were 850, 355, 212, 106, 75, 45 μm purchased from DUAL MFG  and the cumulative distribution curve was predicted referencing the Weibull cumulative probability function as,Q(xk) was obtained by multiplying this particle size frequency  with the weight fraction  as,wi is the weight fraction of particles collected on a sieve i , pj is the frequency of particle at size xj in the sieve i. As a result, the mean particle diameter N is the total number of particles.Imaging analysis for the size distribution was performed on the particles collected on each sieve, respectively. Backlight optical micrographs of the collected particles were taken, and the Image J software was used to calculate the frequency of particle sizes pertinent to each sieve. The overall particle distribution 2D simulation of particle collisions lacking a starch inter-particle layer was performed by assuming the particles moving in the medium as deformable sticky particles. Deformable particles were modeled using polygons in which the vertices were freely jointed, but the area was conserved. Stickiness, that gives rise to particle coalescence after their collision, was included in the simulation via an energy term proportional to the particle surface.The total number of particle collisions with respect to viscosities of the medium was obtained in a 3D setting by assuming no collisions to cause particle coalescence. Therefore, the total number of particles in a controlled 3D space was maintained the same over time. See Supplementary Note 2 for details.Self-propelling Janus particles were fabricated by emulsifying a mixture of 1 ml semicosil-964, 0.3 ml toluene (solvent) and 0.5 g silver-coated ferrite particles in 50 ml gelatinized starch/water medium (10 wt% of starch concentration). The emulsification was performed at room temperature for 30 s using the homogenizer applying a shearing rate of 600 rpm. The resulting vitrified emulsion then underwent a resting period of 3 h to allow the silver-coated ferrite particles to sediment and the polymer resin to cure.Unidirectionally magnetized stretchable particles were fabricated by emulsifying a mixture of 2 ml Eco-Flex 00-30 (two parts mixed at 1:1 by weight) and 0.8 g iron powder in 50 ml gelatinized starch/water medium (10 wt% of starch concentration). The emulsification was performed at room temperature for 1 min using the overhead mixer that applies a shearing rate of 600 rpm. The resulting vitrified emulsion was placed on top of a 50 mm neodymium magnet  to unidirectionally align the iron powders and remained for 3 h to allow the polymer resin to cure. Both Janus particles and unidirectionally magnetized particles were cleaned via the aforementioned procedure that uses amylase enzyme.Self-propelling particles were realized by placing Janus particles in a 100 mm PYREX petri dish, and 30 wt% hydrogen peroxide solution was poured to initiate a reaction that creates oxygen bubbles on the side of the Janus particle surfaces covered with silver-coated ferrite particles. A surfactant Triton X-100 (0.5 wt%) was added to the solution to spur the oxygen bubbles on to be detached from the particle surface. Since the heavier ferrite particles settle and become deposited from the very bottom of polymer droplets, we hypothesize the amount of the ferrite particles required to impart the self-propelling ability will be notably less than what is used in this study (0.5 g per 1 ml resin). The magnetized particles were actuated by placing into a rotating magnetic field.Supplementary informationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5


The strong spatial confinement of a nanocavity plasmonic field has made it possible to visualize the inner structure of a single molecule and even to distinguish its vibrational modes in real space. With such ever-improved spatial resolution, it is anticipated that full vibrational imaging of a molecule could be achieved to reveal molecular structural details. Here we demonstrate full Raman images of individual vibrational modes at the ångström level for a single Mg-porphine molecule, revealing distinct characteristics of each vibrational mode in real space. Furthermore, by exploiting the underlying interference effect and Raman fingerprint database, we propose a new methodology for structural determination, which we have called ‘scanning Raman picoscopy’, to show how such ultrahigh-resolution spectromicroscopic vibrational images can be used to visually assemble the chemical structure of a single molecule through a simple Lego-like building process. The determination of the chemical structure of a molecule is a premier step in chemistry. In the past century, different spectroscopic tools, such as nuclear magnetic resonance  and elec−11 torr) and in liquid-helium cryogenic conditions (∼7 K)  is the confined field distribution function centered at the tip position R0. In the representation of atomic orbital basis, the wavefunctions of the ground and excited state can be described byThe high spatial resolution of SRP images for a specific vibrational mode anoscale ,25,30–32andrespectively. Thus,αϕ(β) is the atomic orbital of the atom α(β). For a realistic field distribution at the ångström level, two possible situations could occur. When the tip is on the top of an atom α, the Raman signal from this atom becomes dominated, as represented by the first term of Equation . Specifically, the well-resolved eight small dots mark the positions of eight C−H bonds, stemming from the out-of-phase destructive interference associated with an anti-symmetric vibration shown on the right of Fig. −1 mode is generated from the in-phase constructive interference between two neighboring C−H bonds associated with a symmetric vibration  stretching vibrations in the range of 2800–3400 cmterature . Upon slg region ,34. The −1 correlated with C = C stretching vibrations. The strong position-dependent spectral features shown in Fig. −1, 1377 cm−1, 1463 cm−1 and 1499 cm−1 in Fig. sp2 carbon atoms involved in C−H stretching vibrations  for visually assembling the chemical structure of a single molecule. It is achieved by combining Raman spectral fingerprints for individual chemical groups with ångström-resolved Raman images and the interference effects involved. The rich spectral data and detailed spatial images of various vibrational modes themselves are already sufficient to provide a panoramic and global view of the molecular structure, which is more comprehensive than the verbal descriptions could give here. The Lego-like building process employed here can be easily generalized with the aid of imaging recognition and machine learning techniques, or by further combination with non-contact AFM and inelastic tunneling probe techniques. The SRP protocol established in this proof-of-principle demonstration can be widely applied for identifying the chemical structure of different materials at the level of chemical bonds.nwz180_Supplemental_FileClick here for additional data file.


In this work, weshow how the structure and intermolecular interactionsaffect the dynamic heterogeneity of aprotic ionic liquids. Using calorimetricdata for 30 ionic samples, we examine the influence of the strengthof van der Waals and Coulombic interactions on dynamic heterogeneity.We show that the dynamic length scale of spatially heterogeneous dynamicsdecreases significantly with decreasing intermolecular distances.Additionally, we assume that the magnitude of the number of dynamicallycorrelated molecules at the liquid–glass transition temperaturecan be treated as an indicator for a dynamical crossover. Such abnormal molecular dynamicsbehavior is often rationalized in terms of correlated motions of theneighboring molecules. Adam and Gibbs introduced the idea of cooperativelyrearrangement regions (CRRs), defined as a group of molecules thatcan change their configurations independently of their surroundings.The sizes of CRRs do not have to be constant in time and/or space,and the dynamics of molecules, which belong to different regions,are not identical.14 Consequently, molecules only a few nanometers away from each othermay have relaxation rates that differ by several orders of magnitude.Hence, molecular dynamics is heterogeneous. These cooperating domains’size increases as the temperature drops, which means that larger andlarger groups of molecules in the supercooled liquid move cooperativelywhen approaching the glassy state. Therefore, the sizes of CRRs areoften considered to play a crucial role in molecular dynamics near Tg.The concept of dynamicheterogeneity of supercooled liquids hasbeen extensively studiedNα, at Tg is in the range of 100–400for van der Waals liquids and H-bonded liquids, 400–600 foroxide glass formers and selenium, and 200–820 for polymers.18 The highest value  was found for poly(vinyl chloride)by Capaccioli et al.18 Surprisingly, sofar, there are no systematic studies of the dynamic heterogeneityof ionic systems. Recent research for protic ionic liquids (PILs)has shown that Nα at Tg for selected hydrochloride salts is smaller than thatof their base counterparts.19 For example,the number of dynamically correlated molecules is equal to 225 forcarvedilol base and Nα = 30 forcarvedilol HCl. To explain these findings, the authors proposed thatlong-range electrostatic interactions induce a small scale of spatiallyheterogeneous dynamics in PILs. Simultaneously, the authors discoveredthat the relative change in PIL’s dynamic heterogeneity andits base depends on the molecular complexity of the salt cation. Hence,it cannot be ruled out that the topological constraints caused bysimpler or more complex anions and cations result in a larger dynamiclength scale of some ionic liquids (ILs). Moreover, since the abovework was limited to protic ionic materials only, we still do not knowwhether the small scale of spatially heterogeneous dynamics is a generalrule for all ion systems or just the unique feature of PILs. Thus,systematic studies are needed to draw a general conclusion about themagnitude of dynamic heterogeneity in ionic liquids.One of the most fundamental questionsin this field is how thestructure and intermolecular interactions affect the dynamic heterogeneityof supercooled liquids. It has been established that the number ofdynamically correlated molecules, Tg, NαD(Tg), the Donth method20 that requires onlycalorimetric data was applied.The mainaim of our work is to systematically study the dynamicheterogeneity in various aprotic ionic liquids. For this purpose,we will determine the number of dynamically correlated molecules atthe liquid–glass transition temperature for aprotic ionic liquids(AILs) with different cation and anion structures and consequentlywith different strengths of electrostatic interactions as well asthe various contributions of van der Waals forces and hydrogen bonds.This will allow us to provide general conclusions about the size ofthe dynamic heterogeneity of these materials. To determine the numberof dynamically correlated molecules at NαD(Tg) decreaseswith the elongation of the carbon chain substituent in the cationor the anion. Thus, the decrease in intermolecular distances due tothe stronger interactions with the increasing number of −CH2– groups induces a decrease in the dynamic length scaleof spatially heterogeneous dynamics near the glass transition. Atthe same time, we noted that while maintaining the same strength ofthe van der Waals interaction ,the obtained NαD(Tg) decreaseswith the increase of the strength of the Coulomb interactions. Additionally,we investigated the evolution of log10 η(T–1) dependence and we observed the strongconnection between the magnitude of the NαD(Tg) value and existence/nonexistence of the intermediatetemperature, Tb (identified with the onsetof complex dynamics). Consequently, we assumed that the magnitudeof NαD(Tg) can be treatedas an indicator for a dynamical crossover.We found that Table S1. The samples were dried anddegassed under low pressure (1 kPa) at temperatures not exceeding373 K.The supplier of the four ILs was Solvionic,whereas the rest was provided by IoLiTec. Note that in some cases,we have used the same batch of materials as in previous works. Fullnames, acronyms, and purities along with water contents (determinedusing the coulometric Karl Fischer method) of the tested ILs are listedin –1 (7 points per decade) with the strain being dependenton temperature and changing from 0.1 to 1000%. The complex viscosityof the studied samples was determined directly from the frequency-independentpart of the log10 η vs angular frequency graph.The relative uncertainty of the reported viscosity measurements ur(η) coming from calibration and temperaturecontrol did not exceed 7%.The viscosity was determinedby means of an ARES G2 rheometer. In the vicinity of liquid–glasstransition, aluminum parallel plates with a diameter of 8 mm wereused. On the other hand, stainless steel geometries with diametersof 25 and 50 mm were used to obtain the viscosity in supercooled andnormal liquid states, respectively. Oscillation shear experimentswere performed in a frequency range from 0.05 to 100 rad·s–1. The samples were cycled at least three times toensure reproducibility and high accuracy. During the experiments,the flow of nitrogen was maintained at a level of 60 mL min–1. Enthalpy and temperature calibrations were performed using indiumand zinc standards.Calorimetricexperiments of the studied ILs were performed by means of MettlerToledo DSC1STAR system equipped with a liquid nitrogen cooling accessoryand an HSS8 ceramic sensor (a heat flux sensor with 120 thermocouples).Each sample with a mass of around 10–20 mg was measured inaluminum crucibles with a 40 μL volume. Prior to the measurement,the samples were annealed for 30 min at 373 K. Temperature ramps involvedcooling to 143 K and then heating to 373 K with a rate of 10 K min4C1pyr][DCA] to 230.8 K for[C4C1im][Cl]. Additionally, two binary mixturesand one ternary mixture were investigated  dependence is a good method to determine the size oftemperature fluctuations.22 Consequently,the number of dynamically correlated particles, NαD(Tg) can be calculated using the following relationM is a molar mass ofthe tested sample, Tg denotes the glasstransition temperature, kB = 1.38 ×10–23 J·K–1 represents theBoltzman constant, NA = 6.02 × 1023 mol–1 means the Avogadro constant, cpglass and cpliquid denote the isobaric heat capacities of glassand liquid at Tg, respectively, and δT is the average temperature fluctuation related to thedynamic glass transition . An excellentdescription of the method with all details and explanations why theabove procedure is suitable for the estimation of Nα at Tg is presentedelsewhere.19One of the most widely used methods for calculating NαD(Tg) values using NαD(Tg) using theDonth method is presented in NαD(Tg) values arefound for [C2C1im][TCM] and [C4C1im][TCM] having an anion with the symmetrical plane structurecausing relatively weak cation–anion interactions due to equivalentchemical competition between same binding sites, i.e., between thethree cyano groups (−CN groups). Moreover, it is almost impossiblefor the tricyanomethanide anion to form H-bonds with the cation.23 Consequently, ILs having an anion [TCM] arecharacterized by exceptionally low viscosity and high conductivity.24 Neves et al.25 reporteda similar range of viscosity and conductivity for other ionic liquidswith cyano-functionalized anions, i.e., thiocyanate-based and dicyanoamide-basedILs, and importantly, one can see that for all these ILs, NαD(Tg) values are relatively large(see NαD(Tg) for cyano-basedILs decreases in the order [TCM]− > [DCA]− > [SCN]−. This sequence is closelyrelated tothe change in the strength of the Coulomb interactions that occurat the bulk liquid. Namely, it is well known that an increase in ionsize reduces the electrostatic attraction. Therefore, we can assumethat the number of correlated molecules decreases with increasingelectrostatic interaction strength, confirming the results obtainedrecently for protic ionic liquids.19 Anotherexciting finding coming from 28 dependenceat Tb (interpreted as the intermediatetemperature) is observed. According to literature data, Tb is identified with the onset of complex dynamics. Thereis a long list of evidence that some qualitative changes occur inthe dynamics of glass-forming systems at Tb.−0.5) to transforms VFT behavior intoa linear dependence on inverse temperature. In this work, we analyzedthe shear viscosity (η) determined by means of an ARES G2 rheometerand representative results together with Stickel analysis are presentedin Tb is 71 ± 10 K higherthan Tg in all cases except for [C2C1im][SCN], where the received Tb is only 31 K higher than the glass transition temperature.Consequently, we determined argesee 1. Morepms at Tb. To demonund for CC1im[TCM]NαD(Tg) value is strongly correlated with the alkyl chain lengthattached to the cation or anion. Namely, based on the results obtainedfor [CnC1im]Cl , [CnC1im][TFSI] , [CnC1im][BF4] , [CnC1im][TCM] , and series, it is obvious that NαD(Tg) clearly decreases with theelongation of the alkyl chain length in the cation or anion . As reported by Vilas et al.,31 theelectrostatic potential decreases initially until a stationary valueat C6 (some authors suggested C4 instead ofC6) is overlapped by the nearly linear van der Waals interactionfunctional , resulting inthe highest overall interaction potential for [C8C1im][TCM]. Since at the same time, [C8C1im][TCM] has the lowest NαD(Tg) value,we can assume that the decrease in intermolecular distances due tothe stronger interactions with the increasing number of −CH2– groups induces the decrease in the dynamic lengthscale of spatially heterogeneous dynamics near the glass transition.In this context, it is worth noting that this statement agrees withthe results reported by Koperwas et al. and Grzybowski et al. Namely,in the mentioned works, the authors observed that reducing the intermoleculardistances as a result of compressing the system also causes its homogenization,i.e., a decrease in Nα. It shouldbe stressed that we do not observe visible changes in NαD(Tg) values around n = 6 for[CnC1im][TCM]  and [CnC1im][NTf2]  when, as was reported inthe literature,33 the investigated ILs begin to form nanoscale aggregates .Additionally, from further inspection of the obtained results,it becomes apparent that the nion see 3. ImportNαD(Tg) is almost independent of the size of thenonaromatic ring,i.e., nearly the same values were obtained for pyridinium-, piperidinium-,and azepanium-based ILs (NαD(Tg) = 31, NαD(Tg) = 27, and NαD(Tg) = 30, respectively) with the same anion (TFSI)and the same alkyl chain length attached to the cation. It shouldbe noted that these NαD(Tg) valuesare one of the smallest among the investigated ILs, and Tb in all these cases was not detected.Another interesting finding from Tb, characterizing a transition from simple dynamics to complex dynamics,is related to the number of dynamically correlated ions. To answerthis question, we analyze the rest of the results. One can see inthe insets of 4C1im][BETI] and [C4C1im][TFSI] with NαD(Tg) values equal to 49.8 and 77.0, respectively, a linear dependencein the whole investigated temperature range is observed, confirminga single-VFT-type behavior. Moreover, for other ILs where the determined NαD(Tg) values are relatively small(<100), one VFT equation may be satisfactorily applied to describethe viscosity data in the whole range. On the other hand, if NαD(Tg) is highenough , wecan detect the change from one VFT to another  series, NαD(Tg) in each case is higher than 100, and in allcases, we observe a dynamic crossover. Notably, for nonionic glassformers where NαD(Tg) is higherthan 100, a dynamic crossover is experimentally observed in thesematerials. Furthermore, the change from one VFT to another seems tobe less visible with decreasing NαD(Tg) value . It turnedout, first, that with increasing the components of the mixture, theparameter NαD(Tg) in relationto [C4C1im][TCM] decreases and, second, thelog10 η(T–1) dependence exhibits clear double VFT behavior for the binary mixture,and the crossover almost disappears entirely for the three-componentmixture  clearly decreases with theelongation of the carbon chain substituent in the cation or anion.Thus, the decrease in intermolecular distances due to the strongerinteractions with the increasing number of −CH2–groups causes a decrease in the dynamic length scale of spatiallyheterogeneous dynamics. Simultaneously, we noted that the obtained NαD(Tg) decreases with the increaseof the electrostatic interactions. Additionally, we observed the connectionbetween the magnitude of the NαD(Tg) value and the existence/nonexistence of the intermediatetemperature Tb in the evolution of viscosity(log10 η(T–1)). This is especially interesting in the context of the physicalmeaning of Tb, which is believed to bea border between complex (T < Tb) and simple (T > Tb) dynamics. Thus, if the number of correlated moleculesis big enough (NαD(Tg) >100,which corresponds to the volume occupied by ionic species equal to∼30 nm3), we can detect that change from simpleto complex dynamics, i.e., two VFT equations with two different setsof fitting parameters are necessary to describe the temperature dependenceof log10 η. Consequently, we assumed thatthe magnitude of NαD(Tg) canbe treated as an indicator for a dynamical crossover.In summary, we obtained the number of dynamically correlated moleculesusing the Donth method for 30 ionic systems (27 pure ionic liquidsand 3 mixtures). Among the investigated ILs, [C