Achillea millefolium EO on the development of common bean (Phaseolus vulgaris L.), is still unknown. Moreover, its effect on N2-fixing bacteria, and in general on soil properties has not been studied yet. A greenhouse trial was set up to evaluate both the influence that Achillea millefolium EO and the inoculation with three different Rhizobium strains have on the bean plant and on the chemical and microbiological properties of an agriculturally used Cambisol. Non-inoculated pots were used as control. Our findings showed a decrease in bacterial colony forming units due to EO application and an increase following the Rhizobium inoculation compared to the control. The EO application decreased soil basal respiration and activities of dehydrogenase, urease, β-glucosidase and acid phosphatase. Such effects were stronger with higher oil concentrations. Moreover, the treatments combining Rhizobium inoculation with EO showed a positive effect on nodulation and plant height. Overall, the combined application of Achillea millefolium EO and rhizobia works as an efficient biocide that could be applied in organic agriculture without hampering the activity of nodule-forming N-fixing bacteria and the development of common bean.Essential oils (EO) of several plant species have the potential to combat plant and fungal diseases. However, the effects of  Enhancing the productivity of agricultural systems while substantially reducing environmental impact is a worldwide responsibility in order to ensure food security for future generations.It is expected that the world population will reach 9.8 billion people by 2050, and 11.2 billion in 2100 according to the United NationsPhaseolus vulgaris L.) is one of the most important legumes in the world2. In areas of Eastern Africa and Latin America, this plant contributes to 65% of the total protein consumed and 32% of the energy intake3. Moreover, it has been estimated that approximately 26 million metric tons of beans were produced worldwide in 20164, being a crucial crop in developing countries5. In developing areas, a sustainable practice to enhance common bean production consists in their inoculation with symbiotic and associative bacteria to enhance plant growth6.Common bean  fixation resulting in better plant developmentRhizobium has been shown to be an environment-friendly alternative to mineral nitrogen fertilizers, due to their positive effects on plant nodulation, biological N-fixation and consequently N-availability. Although the fixed nitrogen is not readily available to the plant, inoculation ensures long-term N-availability in soil, thus, being an ecologically viable option for amelioration of agricultural soils23. In line with this, inoculation of Rhizobium sp. BARIRGm901 in gray terrace soils led to higher nitrogen fixation and yield of soybean (Glycine max)24. Moreover, Rhizobium inoculation does not only affect the plant performance but also the soil microbiota; indeed, Fall et al.25 found that Rhizobium inoculation on a Gum Arabic production area with natural trees of Senegalia senegal (L.) Britton,  led to an increase in soil microbial biomass and respiration as well as mineral nitrogen content; and positive effect on soil microbial functional diversity was also observed25. Numerous studies have examined the influence of plant essential oils with regard to plant protection as biological control agents, either alone or in combination with alternative fungicides. However, there is still scarce information on how soil productivity and plant growth are affected by the application of essential oils alone or in combination with Rhizobium, and if these affect the microbiota in an antagonistic or synergistic way.The use of bacteria from the genus Achillea millefolium essential oil and inoculation with different Rhizobium strains on soil biological properties as well as on the growth of bean (Phaseolus vulgaris L.) and its nutrient content. For this purpose, we determined the effects on soil enzyme activities, soil microbial biomass and respiration, plant biomass and plant nutrient content (N), together with soil nutrient content  and plant root nodulation.The aims of the present study were to investigate the effects of 26, and its main physiochemical properties are shown in Table In the present study, a factorial greenhouse pot experiment was set up in July, 2013 in the Helmholtz Zentrum München - German Research Center for Environmental Health . The soil used was collected on a research farm in the municipality of Scheyern . It was classified as CambisolAchillea millefolium plants were collected from the research campus at Helmholtz Zentrum München - for Environmental Health  in June 2013. Freshly collected leaves were shade-dried at room temperature (22 ± 1 °C) for 10–15 days. Afterwards, the dried leaves were ground into a fine powder with a pestle and mortar. 50 gr of the sample was subjected to hydro distillation for 3 h in 500 mL distilled water using a Clevenger type apparatus for 4 h in order to produce essential oil. After distillation, the EO concentration was determined by gas chromatography-mass spectrometry (GC-MS). Concentrated EO (1500 ppm) were then diluted using distilled water to the following concentrations: 0 ppm: 0 ml EO 100 ml−1, 100 ppm: 6.66 EO 100 ml−1, and 1000 ppm: 66.6 EO ml 100 ml−1.Leaves of Rhizobium leguminosarum biovar phaseoli cultures were obtained from the laboratory stock culture collection and prepared by sub-culturing in three mL of YMA (yeast extract–mannitol agar) and incubated at 28 °C for 5 days. Rhizobial inocula  were incubated at 30 °C for three days in YMA, and were posterior used as material for inoculation in this experiment27. In order to have equal numbers of bacteria as inoculation material, absorbance values at 540 nm wavelength were measured spectrophotometrically. To achieve an equal bacterial density, the lowest number of bacteria after 24 h was taken as a baseline. Where appropriate, bacterial suspensions were diluted to the same absorbance value with sterile physiological saline solution (PSS)27.Achillea millefolium essential oil doses (E0 (control), E1 (100 mg kg−1) and E2 (1000 mg kg−1)) per kg of pot soils with three different Rhizobium inocula and a non-inoculated control . All treatments were done in triplicate, with a total of 36 pots.R0 E0 No inoculation/No essential oil−1)R0 E1 No inoculation/essential oil (100 mg kg−1)R0 E2 No inoculation/essential oil R1 E1 Rhizobium leguminosarum biovar phaseoli F7/essential oil (1000 mg kg−1)R1 E2 Rhizobium leguminosarum biovar phaseoli F83/No essential oilR2 E0 Rhizobium leguminosarum biovar phaseoli F83/essential oil (100 mg kg−1)R2 E1 Rhizobium leguminosarum biovar phaseoli F83/essential oil (1000 mg kg−1)R2 E2 Rhizobium leguminosarum biovar phaseoli Ciat 889/No essential oilR3 E0 Rhizobium leguminosarum biovar phaseoli Ciat 889/essential oil (100 mg kg−1)R3 E1 Rhizobium leguminosarum biovar phaseoli Ciat 889/essential oil (1000 mg kg−1)R3 E2 A total of twelve treatments resulted from combining three different Phaseolus vulgarıs L seeds were surface-sterilized in 5% H2O2 for 15 minutes and rinsed 5 times with sterile distilled water. After the soils were potted, each pot was planted with three sterile seeds. After seedling, the two seedlings showing the weakest growth were removed 7 days after planting. The essential oils were diluted  with 10 ml ethyl alcohol and were slowly surface-sprayed. Seven days after planting, each seedling was inoculated with one mL of log phase bacterial culture with the help of a sterile syringe on the soil surface in the root area. During the 45-day incubation period, soil moisture content was kept at field capacity. The pots were irrigated with water containing bacterial culture suspensions (diluted to 1 × 109 cells in each pot) to 55–60% of the soil water-holding capacity. Soils were maintained at this moisture level by watering them to weight every 2–3 days. After the incubation period, plants were removed from the pots and soil samples were collected and kept at 4 °C prior to analysis.28. Soil cation exchange capacity (CEC) and organic matter (OM) were analyzed as described in Sumner and Miller29 and Nelson and Sommers30 respectively. Soil inorganic nitrogen (N-NH4+ and N-NO3−) was determined in 2 M KCl extracts according to Bremmer and Keeney31. To determine extractable inorganic P, soil samples were extracted with 0.5 M NaHCO3 (pH 8.5) for 30 min, and P was measured following the method proposed by Olsen and Sommers32.Soil electrical conductivity (EC) and pH were determined in 10 g of soil in combination with 25 mL A.d. at a ratio of 1:2.5. Soil texture was analyzed according to Bouyoucos−6 and 10−7, and 100 μL of each sample was then plated on soil extract medium and dextrose-peptone agar for bacteria and fungi, respectively. Bacterial plates were incubated at 30 °C, and fungal ones at 25 °C for a total of seven days33. In the case of bacterial colonies, the average CFUs per gram of oven-dried equivalent (ODE) of field-moist soil was calculated by using an automated colony counter35. Fungal colonies growing in agar were observed under a dissecting microscope. They were then marked and recorded in the surface agar. Total culturable fungi were calculated by viable fungal spore g−1 of oven dry soil36. It has to be addressed that this culture‐dependent method is biased because it generally underestimates the total cell number by a factor of 10–10037.Bacterial and fungal abundance in the soil samples were assessed by determining colony forming units (CFU). One g of soil was extracted in 9 mL phosphate buffered saline (PBS) in a rotation shaker for 30 min. Samples were diluted 1038. Briefly, the CO2 released from 20–25 g moist soil sample previously incubated at 25 ± 1 °C for 24 h was then trapped in 20 mL of 0.5 M NaOH solution. Afterwards, BaCl2 was added to precipitate the CO2 as BaCO3, followed by a titration with 0.5 M HCl.Soil basal respiration (BR) was determined according to Islam and Weil−1 h−1 following the method of Tabatabai39. Soil urease (UE) activity was determined using the steam distillation methods as described by Tabatabai and Bremner40 using urea as substrate and expressed as µg NH4-N g DM−1 2 h−1. β-glucosidase activity (β-Glu) was assayed using β-PNPGLU (p-Nitrophenyl-β-D-glucopyranoside) as substrate and expressed as µg PNP g DM−1 h−1 39. Dehydrogenase (DH) activity was determined with the TTC (triphenyl tetrazolium chloride) method39 and expressed as µg TPF g DM−1 24 h−1.Five soil enzyme activities were measured: acid phosphatase (AcdP) and alkaline phosphatase (AlkP) activities were assessed by using pNPP (para-Nitrophenyl phosphate) as substrate and expressed as µg pNP g DM41.The growth response (plant height), total plant biomass, nodule number, and plant total N-content were measured in this experiment, as these parameters are well-known to show a quick response when plants are inoculated with nodule-forming N-fixing bacteria. Both the aboveground (leaf and stem) and root biomass were collected separately from the experimental pots and washed. Roots were oven-dried at 65 °C for 72 h, while leaves and stems were dried for 48 h to determine their dry matter content. After the plant harvest, the numbers of nodules per plant were determined in the roots. Total plant nitrogen contents were determined by the Kjeldahl method after salicylic-sulfuric acid digestionStatistical analyses were performed using the SPSS 23 Software . Data were analyzed using a two-way analysis of variance (ANOVA). When significant differences were obtained, an analysis with Tukey’s HSD (honestly significant difference) comparison procedure was further performed as a post hoc test. Prior to analysis, data were tested for normality and transformed to meet this condition when necessary. When data did not meet the normality assumption, they were subjected to non-parametric tests for several independent samples  and pair-wise comparisons were performed using the non-parametric Friedman test.Rhizobium strains and the application of different doses of Achillea millefolium essential oil as well as their interaction did not induce significant effects (p > 0.05) on different soil N-pools   the application of E1 induced an increase in the nitrate content, while no such increase was observed for E2.The inoculation of soil with different ) Tables  and 4. D) Tables  and 4. Ip < 0.001), as did the inoculation with different Rhizobium strains (p < 0.001). Moreover, a significant interaction between the application of essential oil and Rhizobium strains (p < 0.001) was also found (Table Rhizobium strains (p < 0.001) was found to affect soil fungal CFUs . A lower reduction was found for higher oil concentrations (E2).The application of essential oil doses to the soils significantly decreased bacterial abundance (Tables nd Table . The bacnd Table . The appUs Table . Each coUs Table . For thep < 0.05). In general, the application of the oil extracts decreased of soil basal respiration   Table .Achillea millefolium L. oil extracts on the activity of the different soil enzyme activities, no effects were observed, except for β-Glu (p < 0.05) for which a reduction was detected compared to the control.Regarding the effects of p < 0.001, for all enzymes), when soil was inoculated with the different Rhizobium strains , while the minimum AcdP activity (10.2 ± 0.5 µg pNP g DM−1 h−1) was observed in the non-inoculated treatment  that showed double the activity in comparison with the other treatments  showed a significant decline in their activity  was observed in the non-inoculated treatment, while the minimum UE activity (34.4 ± 3.7 µg NH4-N g DM−1 2 h−1) was measured in R1 . The maximum β-Glu activity (358 ± 29 µg PNP g DM−1 h−1) was observed in the non-inoculated treatment, while the lowest was found in R3 (188 ± 27 µg PNP g DM−1 h−1)  Fig. . In lineRhizobium inoculation treatments on plant height are displayed in , a significant decrease was observed (Tables p < 0.001). A higher average number of nodules was observed for R2 and R3 , compared to R1 (29.5 nodules) and the non-inoculated control, for which no visible nodules were observed. Moreover, significant interaction between both treatments  was observed for the number of nodules  yielded a higher biomass than the control one (Table −1 respectively) than the non-inoculated control (approx. 1.8 g kg−1).The effects of the application of different in Table . Plant hd Tables  and 5. C44. In fact, the application of Rhizobia and plant extracts in agriculture could be considered as a potential strategy for their application in organically managed and ecologically oriented farming systems. This is also true for the combined effect of Achillea millefolium essential oil and Rhizobium inoculation based on their potential influence on soil chemical, biological and plant growth parameters. Moreover, to our knowledge, there are no studies yet that have considered these factors.The presence of plant growth promoting bacteria (PGPB) in agricultural soils and their dynamics with plants has gained increasing interest among the scientific community thanks to the abilities of PGPB to solubilize nutrientsRhizobium with essential oil, a reduced abundance of both bacterial and fungal CFUs was measured in the present study. However, in the case of the R0E0 treatment, the bacterial CFUs were significantly higher than in the remaining treatments. Oils are known to have inhibitory effects against microbial populations and, in particular essential oil extracted from Achillea millefolium L has shown antimicrobial and antifungal properties15. Although the antifungal effect in the treatments containing oil was significant, such effect was not as strong as that registered for bacteria.For all of the combinations of Rhizobium to suppress both the bacterial and the fungal abundances. However, the specific oil component responsible for this suppression, and the underlying mechanisms behind this effect are not clear yet.In our study, it has been observed that even a small amount of essential oil had a positive effect in combination with 45 also recorded a decrease in the bacterial abundance in cucumber plants after four weeks following inoculation with vesicular-arbuscular mycorrhizal fungi (AMF). Also, a pronounced antibacterial response of oil extract from Achillea millefolium on Staphylococcus species was reported46. Moreover, the inhibitory effect of essential oils on several plant pathogens has been observed in previous studies49. However, other studies reported an enhanced soil metabolism and microbial activity following the application of essential oils52. In addition, it has been demonstrated that small amounts of Lavandula stoechas L. essential oil have a positive effect on mycorrhiza development53. Thus, the utilization of plants containing this essential oil in agriculture can be beneficial due to the natural origin of such oils, and their efficient action against pathogens based on the fact that they contain multiple active compounds, which hampers pathogen resistance development54.In line with our results, where we observed that the inoculation with 2 of the 3 strains led to a reduction in bacterial abundance, Christensen and Jakobsen55. A plausible explanation could be that the application of this type of oils induced a shift in the soil microbial community favouring the appearance of bacterial strains that are resistant and/or capable of catabolizing such components51. Assuming this, we would expect that some biologically active secondary metabolites derived from the extracted essential oil of Achillea millefolium L might be able to alter soil microbial communities and promote the beneficial microbes involved in a rapid colonization of rhizosphere. However, the short duration of our experiment (45 days) made it impossible to confirm such observations.Soil microbial activity, measured as basal respiration in this study, reached higher values when no essential oil was applied for R0 and R1; and decreased with the addition of essential oil except for the R3E2 treatment. Several researchers have documented an enhancement of soil respiration after the application of essential oils and their constituentsrhizobial strains induced a decrease in most of the measured soil enzyme activities, in particular DH, which can be linked to the soil microbial biomass since its activity serves as an indicator for the physiologically active soil microbiota57. Indeed, some essential oils and the individual constituents do not cause an immediate effect on the soil microbiota, but rather seem to require a certain period of time, between 4 and 16 weeks depending on the studies, before increasing soil microbial abundance and basal respiration55. However, it seems that R1 bacteria are more resistant than R2 and R3 bacteria to the effect resulting from the application of Achillea millefolium essential oil. Contrary to AlkP, AcdP activity reached its maximum level following R1 inoculation, while the lowest AcdP activity was observed in the no inoculation treatment. Although alkaline phosphatase is an enzyme produced during microbe-plant interactions58, it showed a positive response to the application of all Rhizobium inoculation and essential oil combinations compared to the no inoculation treatment. Nevertheless, numerous studies have demonstrated that the activity of alkaline phosphatase can remain unchanged after several treatments like those resulting from silvicultural activities59.In this experiment, the inoculation with different 57 that are linked with soil microbial respiration. The abovementioned activity decrease occurred after both inoculation and essential oil treatments. Both treatments also induced a reduction in soil microbial abundance that could explain the decrease of β-Glu activity, which seems to be highly related to the reduction in microbial carbon60. As UE activity was strongly affected by the inoculation treatments used in our study, the decrease in this enzyme activity could be due to nitrogen utilization by bacteria and reduction in soil N available forms61. In addition62, reported that the amendment with different natural products may act as an inhibitor of UE activity.DH activity showed a significant decline depending on the bacterial species. In addition, DH activity which is primarily involved in the carbon cycle has a pivotal role in the enzyme system of all microorganismsRhizobium inoculation and the treatment with essential oil doses neither affected NH4+ N, NO3− N nor total N contents in soils, despite the increased number of root nodules in the inoculated treatments. However, since in the present study the whole plant was collected, the additional N accumulated in nodules was not added to the soil because it is released only during the decomposition of legume biomass. The rotating cropping system considers the release of N in situ either of the whole biomass, or of the roots and nodules63. Another plausible explanation for the non-significant effect could be related to the short duration of the experiment. Nevertheless, it is important to highlight that plants amended with both treatments showed similar NH4+ N, NO3− N, and total N contents than those from the treatments with no essential oil doses. This indicates that the application of these extracts affects soil microbiota in the short term rather than soil nutrient contents.In contrast to our expectations, Rhizobium inoculation in combination with all of the rates of essential oil showed a positive response in terms of nodule number and plant height. Nevertheless, inoculating common bean and amending with essential oil did not result in a significant increase in plant biomass and plant N content. Previous studies have reported that Rhizobia are considered a main agent for N fixation in plants, root nodulation and crop growth66. Boddey et al.67 found that inoculation of Cowpea with the Rhizobium strain BR 3299 significantly increased grain yield. Furthermore, as stated in Fan et al.68, inoculation of tomato plants with Plant Growth Promoting Rhizobacteria enhanced N and P uptake in the shoots. Contrary to these authors, and in line with our observations, Chekanai et al.69 observed that inoculation with Rhizobium did not induce a positive response in plant biomass or grain yield in common bean (Phaseolus vulgaris L). However, it is known that essential oils are proven to be a thousand times less toxic than standard insecticides, and many of their constituents are not persistent in freshwater and can be metabolized by soil-borne microorganisms10.We observed that Achillea millefolium essential oils alone or in combination with Rhizobium bacteria had an influence on soil productivity and the development of common bean (Phaseolus vulgaris L.). The biocide effect of the application of these essential oils was reflected in a decrease in bacterial and fungal CFUs as well as in basal respiration and soil enzymes activities. Moreover, such effects were stronger with higher oil concentrations. However, the applied essential oil did not seem to harm the effectivity of the used Rhizobium strains, in fact, its joint application increased the number of nodules and the plant height in the studied bean plants. Such effects were not accompanied by an increase in neither inorganic nor total N content in soil, which could be due to the duration of the study. In order to corroborate the positive results of this study, it would be necessary to perform further long-term studies under field conditions to determine the upscale application of Achillea millefolium essential oils, and their potential as additives suitable for organic farming and biologically managed soils.In summary, we conclude that the application of


Assessment of van der Waals (VDW) interactions is fundamental to all of the central quest of structure that regulates the biological function. VDW interactions contributing to intramolecular weak hydrogen bonding are regarded as an important force to regulate the thermal stimuli-sensitive function of sulfobetaine methacrylate, DMAPS. We present here the conversion from the thermal-motion form at room temperature to the weak-hydrogen-bonded form against thermal motion as a terahertz spectral change with a definite isosbestic point from an absorption peak of one form to the other. Vibrational absorptions are used as a probe for assessing VDW interactions in conjunction with highly reliable and well-established density functional theory (DFT) calculations for analysis. Complicated spectral features and uncertain conformations of DMAPS in the amorphous state are clearly resolved under the polarizable continuum model and the dispersion correction for the pure DFT calculations. The VDW interaction named in honour of van der Waals, who first introduced attractive interactions between neutral molecules in his equation of state, is a dispersion interaction of pure quantum physical origin14. The VDW force is well known as attractive at smaller molecular separations, while the sign of the VDW force can be changed from attractive to repulsive by suitable choice of interacting materials immersed in a fluid at larger distances15. The VDW forces are very weak and easily disturbed by the thermal energy around room temperature. In biological systems that function at room temperature, VDW interactions provide fantastic characteristics such as fuzzy control in the function of biomolecules. It is significant to directly assess the VDW interactions. How do we determine the efficiency of a VDW interaction? The influence of curvature strain and VDW force on the interlayer vibrational mode of WS2 nanotubes was reported as a redshift by 2.5 cm−1 with confocal micro-Raman spectroscopy16. The vibration mode is a promising probe for assessing VDW interactions and can be used in various materials, including biological systems. In particular, terahertz (THz) vibration is powerful for detecting weak interactions19. Combined with highly reliable and well-established density functional theory (DFT) calculations for the analysis of vibrational absorption spectra, the effect of dispersive interactions can be directly revealed by the observation of vibrational spectral change at low temperature where thermal disturbance is suppressed and the VDW interaction is effective against thermal motion.The van der Waals (VDW) interaction has become a hot topic in recent years as a constructing force of promising materials such as the VDW heterostructure24, and we have investigated several biocompatible nanoparticulate drug delivery systems26. The biocompatibility and the thermal stimuli-responsive function of polysulfobetaines is stemmed from the zwitterionic sulfobetaine monomer. Betaine is a specific type of electrically neutral zwitterion that contains a pair of cationic functional group bearing no hydrogen and anionic functional group at non-adjacent position in the molecule. It is noteworthy that betaine is in an important position to explore the correlation between the structure and function in biological systems, because the special zwitterionic structure of betaine is biomimic of fundamental biomolecules such as amino acids, proteins, and phospholipids.The polymerized sulfobetaine methacrylate exhibits a thermal stimuli-responsive function where intramolecular VDW interactions have a decisive role. A sulfobetaine methacrylate, DMAPS (3-[dimethyl-(2-methacryloyloxyethyl) ammonium] propane sulfonate) studied here, is an electrically neutral zwitterionic betaine monomer used in the synthesis of biocompatible polysulfobetaines. The development of biocompatible nanoparticles using zwitterionic polymers is increasingly demanded27. The molecular size of DMAPS is small enough to calculate with reasonable accuracy and sufficient level. DMAPS contains intramolecular weak hydrogen bonds predominantly governed by dispersion interactions such as the VDW force, and the formation of the intramolecular weak hydrogen bonds at low temperature is expected to affect the THz vibrations. Amorphous samples are more informative from the biological point of view and the THz spectroscopic study of amorphous samples is desired. However, the study of amorphous samples is lagging behind. A small molecule in an amorphous state gives no spectral feature in the THz spectral region, although a molecule in a crystalline state gives THz peaks28. Early THz spectroscopic studies of proteins provided THz peaks even in noncrystalline states29, but the peak assignment of proteins is difficult due to the large molecular size. Another issue in the THz spectroscopic study of amorphous system is the treatment of surrounding molecules in the calculation for peak assignments. We have studied intermolecular weak hydrogen bonding of crystalline compounds using THz spectroscopy with DFT calculations33 and synchrotron FTIR microspectroscopy34. Nowadays, THz spectroscopic studies are well established for crystalline systems including microcrystalline powder samples and THz peaks due to intermolecular weak hydrogen bonding networks are obtained as coupled with intermolecular translation and rotation modes. THz peaks are very sensitive to the environment of respective molecule, and even the assignment of intramolecular modes in crystalline compounds requires periodic solid-state calculations that include the effects of surrounding molecules in crystals.THz peaks from the methacryloyl moiety of DMAPS are predicted to be observed even in the amorphous state35, and the dispersion forces that contribute mainly to the weak hydrogen bond are one of three interactions  contributing to hydrogen bonding. Dispersive interactions, such as VDW interactions and weak hydrogen bonds, are easily disturbed by thermal energy near room temperature  to strong  depending on strength−1 was measured by synchrotron FTIR microspectroscopy to investigate the behaviour at low temperature where VDW interactions become effective. The overall spectral profile hardly changes upon cooling from room temperature to 4 K, except for peak sharpening and a slight peak shift to high frequency. The spectra from 140 to 700 cm−1 at 150 K and 4 K are shown in Fig. −1. One peak at 150 K splits in two at 307 and 314 cm−1 at 4 K  to 150 K (12.6 cm−1)  spectral profile of DMAPS in the range from 140 to 700 cm−1 where the temperature-dependent peak splitting was experimentally observed in Fig. 36 with a dielectric constant of 8.33 excellently reproduces four peaks around 300 cm−1 ; 286, 301, 314, and 331 cm−1 . The discrepancy between the calculated frequency and the experimental value of Fig. −1) in the range of 140–250 cm−1, while the deviation of the calculated value from experimental result in the range of 250–700 cm−1 is relatively large and always negative (up to 32 cm−1)  exhibits the higher temperature spectrum, because the increase of temperature raises the dielectric constant due to the higher mobility of molecules caused by the cleavage of hydrogen bond at higher temperatures.Figure −1 (N) changes from 5 to 3 as the dielectric constant increases  and 2-pentanone (ε = 15.20) are selected as solvents for the low-temperature model below 100 K and high-temperature model above 150 K, respectively.The number of peaks near 300 cmε, under the PCM model at the B3LYP/aug-cc-pVTZ level with dispersion correction. Among 27 conformations for atoms 1–6 in Fig. ε = 8.33) and high-temperature model (ε = 15.20) are the same 37. The distance gradually increases and its bond strength weakens as the dielectric constant increases and the O atom accepter basicity decreases. At the highest three ε′s where N = 3, the distance is much more than VDW contact (>2.79 Å)38 and no hydrogen bonding exists. At the middle three ε′s where N = 4, HB1 gives a constant value (2.77 Å) suggesting VDW contacts probably due to quantum fluctuations. In that sense, HB1 at the middle three ε′s is a weak hydrogen bond at the VDW limit. For all dielectric constants, HB2 gives a typical O…H distance between the CH donor and the > S = O accepter (2.38~2.57 Å)37, and the permittivity dependence is the same as HB1 at the lowest three ε′s. Even at the middle three ε′s where N = 4, the HB3 distance is greater than 2.79 Å and no hydrogen bonds already exist in this dielectric constant range.Full geometry optimization was performed for the conformer giving the best-fit FIR spectrum to the experimental 4 K result, using a different dielectric constant, −1) shows a clearer temperature-dependent spectral change , and the stronger peak shifts remarkably to higher frequency  (disrotation of one methyl group and N(MeR1R2) group) and τ2 (-CH3) (corotation of two methyl groups on a nitrogen atom)  and high-temperature model (ε = 15.20). The strength of HB2 gradually decreases as the dielectric constant increases. On the other hand, HB1 makes weak hydrogen bond at the VDW limit in low-temperature model, but not in high-temperature model. In low-temperature mode (ε = 8.33), the frequency and intensity of τ1 (-CH3) and τ2 (-CH3) increase due to the increase of HB2 bond strength. Therefore, totally four peaks including τ1 (-CH3) and τ2 (-CH3) were observed below 100 K. Mode τ2 (-CH3) includes movement in the HB1 bonding direction, but not τ1 (-CH3). The cleavage of HB1 at ε = 15.20 (high-temperature model) allows the O atom of the SO3 group to move without constraint from the HB1 bond, and only the intensity of τ2 (-CH3) increases. Thus, totally three peaks were observed above 150 K.The vibrational modes of DMAPS showing a temperature-dependent spectral changes in the FIR and THz regions are examined. The peaks at 307 and 314 cmom) Fig. . As showν1, ν2, and ν3 in Fig. ν2 and ν3, contain stretching vibrations of the HB1 hydrogen bond. As shown in Table ε = 8.33 and not at ε = 15.20. Thus, with the formation of HB1 hydrogen bonding at ε = 8.33, the frequencies of ν2 and ν3 increase.Three peaks observed in the THz spectrum are assigned to three torsion modes  that can attain a consistently accurate description of VDW interactions. We found a very weak intramolecular hydrogen bond at the VDW limit formed below 100 K, and the contact distance is calculated to be 2.77 Å. The formation of very weak hydrogen bonds critically causes a change in the number of peaks around 300 cmDMAPS (3-[dimethyl (2-methacryloyloxyethyl) ammonium] propane sulfonate) was purchased from Sigma-Aldrich  and measured in both synchrotron FTIR microspectroscopy and terahertz time-domain spectroscopy (THz-TDS) without further purification.−1 were collected with a resolution of 2 cm−1 at an infrared beamline BL43IR of the SPring-8 synchrotron radiation (SR) facility . An FTIR (BRUKER IFS120HR) spectrometer was used with IR-SR as an infrared source. The powder sample dissolved in desalinated water was dropped on a silicon substrate and dried, then inserted into the cryostat and evacuated. The spectrum was the same, even when the powder sample was placed directly on a silicon substrate without dissolving in desalinated water and was inserted into the cryostat and measured. By evacuation it is estimated that the water disappears completely during the measurement. The frequency of each peak in Figs. Synchrotron FTIR spectra in the range of 140–700 cm−1 were measured with a Tochigi Nikon RT-20000 THz-TDS with a resolution of 0.8 cm−1 at 11–296 K. Powder samples were placed directly on the sample holder with Kapton tape on both sides. We confirmed that the Kapton tape has no absorption in the THz spectral region.THz spectra from 20 to 60 cm43. We utilized a hybrid Becke-type three-parameter exchange functional44 paired with the gradient-corrected Lee, Yang, and Parr correlation functional (B3LYP)46 and the aug-cc-pVTZ basis set51. The Petersson-Frisch dispersion model from the APDF functional was used for dispersion correction52. This model provides comparable weak interaction accuracy to that of CCSD(T)/aug-cc-pVTZ, which can attain a consistently accurate description of VDW interactions. The geometric parameters were fully optimized, and the optimized structures were confirmed to have no imaginary frequency. To select stable conformers, the Gibbs free energy at 298 K was calculated with highly accurate CBS-Q  radii53 scaled by a factor of 1.1. The scaling factor depends on solvent, solute36, and the definition of atomic radii55. Examining the effects of the scaling factors, we found that the results with factor 1.1 fit well to the FIR spectrum at 4 K and can account for temperature-dependent spectral changes. To obtain the reliable result for very low frequency as in THz region, a superfine integration grid was used.To analyse the vibrational absorption spectrum of a fully amorphous powder sample, DFT calculations of gas-phase molecules surrounded by themselves were performed with the Gaussian 09 and 16 software packages+–O− connection at the B3LYP/cc-pVTZ + ZPE level with dispersion correction 56. We have obtained 24 conformations, starting from 27 conformations of methacryloyl moiety with a transoid -C(=O)-C(CH3=CH2) at the terminal.We investigated all possible conformations of the methacryloyl moiety of the DMAPS molecule for the propane sulfonate moiety in the ring form at the B3LYP/cc-pVTZ to elucidate the conformation of DMAPS in the amorphous state studied here (see Supplementary Fig. Assessment of the VDW interaction converting DMAPS from the thermal-motion form to the hydrogen-bonded form


Angular-momentum nanometrology with a low crosstalk of less than −20 dB is achieved. This compact high-precision angular-momentum nanometrology opens an unprecedented opportunity for on-chip manipulation of optical angular momentum for high-capacity information processing, ultrasensitive molecular sensing, and ultracompact multi-functional optoelectronic devices.Complementary metal–oxide–semiconductor (CMOS) technology has provided a highly sensitive detection platform for high-resolution optical imaging, sensing and metrology. Although the detection of optical beams carrying angular momentum have been explored with nanophotonic methods, the metrology of optical angular momentum has been limited to bulk optics. We demonstrate angular-momentum nanometrology through the spatial displacement engineering of plasmonic angular momentum modes in a CMOS-compatible plasmonic topological insulator material. The generation and propagation of surface plasmon polaritons on the surface of an ultrathin topological insulator Sb Multiplexed vortex light beams are promising for optical communications, but efficient mode sorting is so far limited to bulk optics. Here the authors develop a scalable vortex beam sorter that uses a plasmonic topological insulator structure to spatially separate the modes to resolve them on a standard CMOS detector. Its unique capability to support photonic devices with high-spatial and -spectral resolution has allowed the detection of optical signals in different physical dimensions. The orbital angular momentum (OAM) of light carried by a helical wavefront with a physically unbounded set of spatial modes has emerged as a physically orthogonal dimension of light5. Although the generation and measurement of the OAM of light have recently been exploited by nanophotonic methods10, the separation of OAM modes has been limited to bulk optics that accomplish the linear displacement of OAM-carrying beams12, due to the lack of OAM-dispersive materials or detectors at a nanoscale.Optical imaging, sensing and metrology have been significantly advanced with the development of complementary metal–oxide–semiconductor (CMOS) technology14. As such, nanoplasmonic manipulation and multiplexing of the OAM of light have recently been explored for widespread applications ranging from on-chip optical signal processing15 and high-definition displays17 to high-precision optical micromanipulation18 and highly sensitive chiral molecular detection19. However, observation of linearly OAM-dispersive plasmonic structures is restricted to the characterization by a near-field scanning optical microscopy20, due to the absence of an outcoupling mechanism for the transmission of surface plasmons to far-field detectors, which is essential for on-chip OAM nanometrology devices.Plasmonics has merged high-capacity photonics and electronics at nanoscale dimensions23. Such an ultrabroad operation bandwidth of plasmonics in CMOS-compatible topological insulator materials is highly desirable for integrated photonic detectors, although it is generally not available in conventional noble metals26. To date, however, the surface plasmon polaritons (SPPs) have not been experimentally achieved in topological insulator materials at visible frequencies.On the other hand, topological insulator materials, with a three-dimensional high-index insulating bulk state and a topologically protected two-dimensional metallic surface state, have recently been shown to support localized surface plasmon resonances in an ultrabroad frequency band ranging from the ultraviolet to terahertz frequencies2Te3 thin film exhibit superior plasmonic figures of merit as compared to the noble metal gold in the ultraviolet-visible spectral region. Applying the superior plasmonic effect in an ultrathin Sb2Te3 film with a thickness of 100 nm to the linear displacement engineering of OAM-carrying plasmonic modes at the nanoscale, we demonstrate for the first time a CMOS-integratable OAM nanometrology device with a low modal crosstalk of < −20 dB. Our demonstrated ultrathin OAM nanometrology device with a small footprint of 13.4 µm by 10.6 µm may open exciting avenues for the future development of ultrathin functional optoelectronics.Here, we demonstrate a concept of CMOS-integratable OAM nanometrology in an ultrathin topological insulator film, based on the linear displacement engineering of plasmonic angular momentum fields at the nanoscale. Our dispersion analysis suggests that the SPPs in the topological insulator Sb28 and shifted semi-circular nanostructures17 were demonstrated to transfer their geometry-dependent geometrical topological charges  to plasmonic angular momentum modes. As such, an incident optical beam with the spin angular momentum, SAM (s), and the OAM (l0) can be converted to a plasmonic angular momentum field with the total angular momentum (L) mode of L = s + l0 + lS, governed by the angular momentum conservation in the SPPs coupling. A key enabling aspect of the OAM nanometrology is to design plasmonic nanostructures with different GTCs (lS) to couple optical beams with different angular momentum modes (l0 + s) into linearly displaced plasmonic fields. Here we use spatially shifted semi-circular nanogrooves to achieve a linear spatial displacement of plasmonic angular momentum modes. Owing to the geometric phase originated from the spin–orbit coupling in the SPPs excitation, unidirectional shifting one half of a circular nanogroove with respect to the other half along the breaking line  can be utilized to convert optical beams with different SAM and OAM modes (s + l0 = −lS) into a solid plasmonic focus at the middle location of the displaced semi-circular nanogrooves, which is governed by the total angular momentum conservation (L = s + l0 + lS = 0). Figure s + l0) ranging from −6 to 6 on semi-circular nanogrooves with shift-dependent GTCs (lS) ranging from +6 to −6, respectively, which reveals the linear displacement of angular momentum modes at the nanoscale. To directly measure the angular momentum-dependent spatial shift in the far-field region, angular momentum mode-sorting nanoapertures are allocated in the middle of the spatial shifts to couple out plasmonic angular momentum fields, laying the physical foundation for OAM nanometrology.The design principle and the schematic of CMOS-integratable OAM nanometrology are illustrated in Fig. ine Fig.  allows tfts Fig. . As an e2Te3 thin film. Subsequently, the SPPs fields are coupled out by mode-sorting circular nanoapertures drilled through the thin film, leading to OAM-dependent linear spatial displacement in the detection plane of a CMOS camera .The schematic design of CMOS-integratable OAM nanometrology is given in Fig. 2Te3, we experimentally measured the optical constants of the Sb2Te3 material using the ellipsometry method over a broad wavelength range from 300 to 700 nm. The conductive surface layer of the Sb2Te3 thin film features a thickness of 1.5 nm which was obtained from an ellipsometry fitting model. Specifically, B-spline and Tauc–Lorenz models were used to determine the permittivity of the surface and the bulk layer of the Sb2Te3 thin film, respectively29. The spectra of the real part of the permittivity of both the surface state and the bulk state of the Sb2Te3 thin film are shown in Fig. 2Te3 thin film remains negative in the whole measured wavelength range, which indicates that both the bulk state and surface state of the topological insulator Sb2Te3 can support the SPPs in principle. Moreover, the bulk dielectric permittivity of the Sb2Te3 thin film implies a large refractive index at optical frequencies, which might result from the complex electronic structures of topological insulator materials, including indirect gap, the density of state near the Fermi surface, and electron–hole interactions31. Figure 2Te3 thin film and air, which is excited at a visible wavelength of 640 nm by using a nanograting coupler.To perform the dispersion analysis of the SPPs in the topological insulator material of Sbprop) and degree of confinement (PFOMconf) are characterized26. The plasmonic figures of merit for the propagation length and the degree of confinement are defined as k|| and k⊥ are the transverse and longitudinal wave-vectors on the dielectric (air) side of the Sb2Te3/air interface, respectively, and λ0 is the excitation wavelength of light. To effectively evaluate the figures of merit of the Sb2Te3 thin film, its permittivity was experimentally retrieved from spectroscopic ellipsometry data, without modelling it as a multi-layered structure22  from a circular nanoaperture waveguide was numerically characterized as a function of the radius of the nanoaperture  ranging from 150 to 200 nm, the transmittance of total angular momentum modes of L = ±1 and ±2 is of more than two orders of magnitude larger than other higher order modes. As such, here we regard the total angular momentum mode of L = ± 1 as the desired one to be preferably supported by the mode-sorting nanoaperture with a radius of 150 nm. Notably, the generalized OAM nanometrology concept could be extended to sort different combinations of angular momentum modes in a sequential order, with the use of nanoring slit waveguides which exhibit single angular momentum mode sensitivity  the coupled SPPs fields are selectively coupled out by mode-sorting nanoapertures with r = 150 nm at different locations. Without loss of generality, we use four semi-circular nanogrooves with the GTC (lS) of −6, −2, +2, +6 to support the transmittance of optical beams with l0 = + 6, s = +1 (AM1), l0 = + 2, s = +1 (AM2), l0 = −2, s = −1 (AM3), l0 = −6, s = −1 (AM4), respectively.Combing the features of the spatial shift-dependent GTCs by the nanogrooves and of the mode-sorting sensitivity by nanoapertures, a physical approach for CMOS-integratable OAM nanometrology can be developed. The key steps of this approach are: (1) optical beams with different SAM and OAM modes are converted into spatially separated SPPs fields with an identical total angular momentum mode of 2Te3 thin film with a thickness of 100 nm  through the designed OAM nanometrology device leads to the spatially shifted optical transmission of the four AM beams  in the far-field region 3 + 3[(CH3)3Si]2Te → Sb2Te3 + 6(CH3)3Si-OC2H5. The injection/purge times for the precursors of Sb(OC2H5)3 and 3[(CH3)3Si]2Te were 0.75 s/9 s and 0.1 s/9 s, respectively. An Ar gas booster was used on the cylinder to promote the gasification process of Sb(OC2H5)3 precursor. The chamber temperature was kept at 70 °C and the chamber vacuum was 2 × 10−2 Torr. The growth rate was 0.01 nm per cycle. X-ray diffraction (XRD) and Raman spectrum confirms that the Sb2Te3 thin films are thin film is highly crystalline and grow along the c-axis.The 100 nm-thick Sb2Te3 thin film using the focus ion beam (FIB) lithography method. With the use of the ion ablation, the periodic nanogrooves in the plasmonic lens and semi-circular nanogrooves and nanoapertures in the OAM nanometrology device were patterned in the Sb2Te3 thin film. In the plasmonic lens, the inner nanogroove has a radius of 5.4 μm. The groove width and the period of the nanogrooves were 250 and 605 nm, respectively. In the OAM nanometrology device, the nanoapertures were drilled through the Sb2Te3 thin film and the shallow nanogrooves were with a depth of 50 nm. The transverse shift of semi-circular nanogrooves is multiple times of the SPPs wavelength (532 nm) at a visible wavelength of 561 nm.We fabricated the ultrathin plasmonic lens and the OAM nanometrology device on a 100 nm-thick Sb2Te3 thin film were simulated using the COMSOL Multiphysics. The optical constants of the Sb2Te3 thin film in Supplementary Figure The plasmonic lens and the OAM nanometrology in the SbL) from the Sb2Te3 thin film by sweeping the radius of the nanoaperture. This model is consisting of a shallow circular nanogroove with a thickness of 50 nm, a width of 250 nm, and an inner radius of 2 µm, as well as a drilled-through nanoaperture at the geometrical centre of the circular nanogroove with a dynamically swept radius. The grooved and ungrooved surface sides  of the Sb2Te3 thin film were further placed onto different dielectric layers with a refractive index of 1 (air) and 1.5 (glass) and the same thickness of 1 µm, respectively. The non-diffracting OAM-carrying chiral beams (SAM) with doughnut-shaped intensity distributions are then adopted as incident beams. The SPPs are launched by illuminating the doughnut-shaped OAM-carrying chiral beams on the nanogrooves.Specifically, to numerically investigate the mode-sorting sensitivity by a nanoaperture, a COMSOL model was built to characterize the optical transmission of different angular momentum modes (2Te3 thin film, wherein new nanogrooves feature a modified inner radius of 5.4 µm. Multiple drilled-through circular nanoapertures were placed at the middle location of the spatially displaced semi-circular nanogrooves with a fixed radius of 150 nm.In addition, to numerically characterize the OAM nanometrology chip, the above COMSOL model was modified by replacing the shallow circular nanogroove with spatially displaced semi-circular nanogrooves on the Sb−4, which has enabled OAM nanometrology signals to be faithfully detected by a CMOS detector under a moderate experimental condition with the measured incident power of 1.25 µW in front of the OAM nanometrology chip.An optical setup was used to measure the OAM-dependent spatially shifted optical signals in the far-field region. This optical system mainly consisted of a 561 nm CW-laser, a linear polarizer, an SLM for the OAM generation in the free space, a QWP for the SAM generation, and a CMOS camera. The schematic diagram is presented in Supplementary Fig. Supplementary Information


In human skin the junction between epidermis and dermis undulates, the width and depth of the undulations varying with age and disease. When primary human epidermal keratinocytes are seeded on collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the epidermal-dermal interface, the stem cells become patterned by 24 h, resembling their organisation in living skin. We found that cell density and nuclear height were higher at the base than the tips of the PDMS features. Cells on the tips not only expressed higher levels of the stem cell marker β1 integrin but also had elevated E-cadherin, Desmoglein 3 and F-actin than cells at the base. In contrast, levels of the transcriptional cofactor MAL were higher at the base. AFM measurements established that the Young’s modulus of cells on the tips was lower than on the base or cells on flat substrates. The differences in cell stiffness were dependent on Rho kinase activity and intercellular adhesion. On flat substrates the Young’s modulus of calcium-dependent intercellular junctions was higher than that of the cell body, again dependent on Rho kinase. Cell patterning was influenced by the angle of the slope on undulating substrates. Our observations are consistent with the concept that epidermal stem cell patterning is dependent on mechanical forces exerted at intercellular junctions in response to undulations in the epidermal-dermal interface.In human skin the epidermal-dermal junction undulates and epidermal stem cells are patterned according to their position. We previously created collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the undulations and provide sufficient topographical information for stem cells to cluster on the tips. Here we show that the stiffness of cells on the tips is lower than cells on the base. The differences in cell stiffness depend on Rho kinase activity and intercellular adhesion. We propose that epidermal stem cell patterning is determined by mechanical forces exerted at intercellular junctions in response to the slope of the undulations. In humans, the interface between the epidermis and dermis is not flat but undulates The importance of physical parameters has been explored by seeding individual epidermal cells (keratinocytes) on ECM-coated micro-patterned islands. Restricting keratinocyte spreading on 20 μm diameter circular islands triggers terminal differentiation whereas cells on 50 μm diameter islands remain spread and do not differentiate Several of the signal transduction pathways that regulate keratinocyte differentiation in response to physical cues have been identified In human epidermis the cells in the basal layer are patterned, with stem cells expressing highest levels of β1 integrin clustered where the basal layer comes closest to the skin surface. We are able to mimic the undulations by creating collagen-coated PDMS substrates and have shown that the topography that most closely resembles healthy human skin induces stem cell clustering, with nuclear YAP, on the tips Although ECM adhesion is known to regulate both the spatial location and the differentiation of keratinocytes in vivo and in vitro 22.1−1 rat tail type 1 collagen, from Becton Dickinson) by adsorption for 2 h at 37 °C.Patterned and flat polydimethylsiloxane (PDMS) substrates were prepared as previously described 2.2−4 M adenine, 10% fetal calf serum (FCS), 0.5 μg mL−1 hydrocortisone, 5 μg mL−1 insulin, 10−10 M cholera toxin and 10 ng mL−1 epidermal growth factor). Low calcium complete FAD contained calcium-free F12 and DMEM supplemented with Chelex 100 (BioRad)-treated FCS Primary human keratinocytes (strain km) were cultured on mitotically inactivated J2 3T3 cells in complete FAD medium , 1.8 × 102. After 45 min at 37 °C non-adherent cells were removed and the substrates were transferred to 12-well plates containing J2 3T3 feeders at a density of 20,000 cells per cm2. In some experiments cells were treated with Rho kinase (ROCK) pathway inhibitor Y-27632 (Life Science Enzo) at a final concentration of 10 μM.Collagen-coated patterned and flat PDMS substrates were used to culture keratinocytes at a seeding density of 150,000 per cm2.3PDMS substrates seeded with keratinocytes were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 h at room temperature. Samples were washed for 2 × 10 min in phosphate buffer. The samples were dehydrated with a gradient of 30%, 50%, 70%, 90% and 3 × 100% ethanol for 15 min at each step. Samples were then dried using hexamethyldisilazane:ethanol (1:1) for 5 min, mounted on an aluminum stub and sputter gold coated (4 nm thickness). Image acquisition was performed using a desktop SEM (JEOL NeoScope JCM 6000Plus).2.4Cells seeded on PDMS substrates were fixed in 4%PFA for 15 min followed by three washes in PBS. Permeabilisation and blocking (PB) buffer  was added and incubated at room temperature for 1 h. Substrates were incubated in primary antibodies for 1 h at room temperature. Samples were washed three times in PBS and incubated with secondary antibodies at room temperature for 1 h. Samples were washed three times in PBS and then mounted on glass slides in Prolong antifade mounting reagent (Life technologies) containing DAPI. Samples were imaged using a Nikon A1 upright confocal microscope with a 20× objective. Images are presented as maximum intensity projections.Primary antibodies used were as follows: anti-β1 integrin mouse monoclonal , anti-involucrin rabbit polyclonal , anti-E-Cadherin mouse monoclonal , anti-Desmoglein 3 mouse monoclonal , anti-MAL rabbit polyclonal , anti-Ki67 rabbit polyclonal , and anti-YAP mouse monoclonal . Alexa Fluor™ 488 Phalloidin  was also used. Secondary antibodies of the appropriate species were conjugated to Alexa Fluor™ 488, 555 and 647 (ThermoFisher Scientific).2.52 with 256 (=16 × 16) or 1024 (=32 × 32) force-separation measurement acquired. Each force-separation measurement was acquired with a maximum ramp size of 6 µm and ramp rate 0.5 Hz, up to a maximum force of 2 nN. Analysis of force-volume scans to obtain the Young’s modulus maps was performed with Nanoscope Analysis software (Bruker) using the linearized Sneddon conical model over the force range 0.4–1.6 nN and assuming a Poisson’s ratio of 0.5.AFM synchronised with optical microscopy  was used to assess the biomechanical properties of the cells. Keratinocytes were seeded on substrates 24 h prior to analysis and measurements were conducted at 37 °C in complete FAD medium. Optical microscopy was used to localise the AFM tip on the top or base of PDMS topographical features. Silicon nitride MLCT cantilevers (Bruker) with nominal spring constants of 0.02 N/m  and 0.6 N/m  were used. Both cantilever types have pyramidal tips with effective semi-included angles of 18° and a nominal radius of curvature of 20 nm. The size for each force-volume scan was selected in the range of 15–50 μm2.6Keratinocytes were seeded on collagen-coated PDMS substrates 24 h prior to live imaging. In some experiments, keratinocytes were infected with lentiviral vectors encoding GFP (pLenti-CMV GFP puro (658-5), gift from Eric Campeau, Addgene plasmid # 17448,) and mCherry . Keratinocytes were transduced with lentiviral plasmids carrying GFP and mCherry reporters 2 days prior to live imaging. A JuLI™ Stage Real-Time Cell History Recorder device was used to acquire images from live keratinocytes. Images were captured with an interval time of 1–2 h and videos were created using JuLI™ Stage edit software.2.7t-test and presented as the mean ± standard deviation. The number of replicate experiments is reported in the figure legends.Prism7 GraphPad software was used to generate graphs and perform statistical analysis. All data were analysed by unpaired or paired ANOVA or paired The immunofluorescence labeled images were processed by imageJ and presented as maximum intensity projections. Using imageJ, cells labelled with DAPI, Ki67, involucrin or other markers were quantified and presented as a percentage of positive cells. Nuclear height and volume were measured using the 3D object counter plugin.33.1We previously generated a set of 9 PDMS substrates (including one flat control substrate) by using three different photomasks and three different UV exposure times 3.2To examine the evolution of cell patterning on the undulating substrates, keratinocytes were cultured on the collagen-coated patterned and flat PDMS substrates for 1 h, 4 h, 24 h and 4 days . β1 inteKeratinocyte motility has been reported to distinguish stem cells from differentiated cells Initially cells were observed moving on the base and tips of the substrates; however, when cells reached confluence movement largely ceased. Prior to confluence GFP and mCherry labeled keratinocytes were observed not only moving relative to one another but also sliding over one another, indicating that once cells have differentiated and left the basal layer they can translocate from their original position . By loweThese results suggest that patterning is achieved by cues provided by the topography of the culture substrate rather than by cell sorting due to cell migration.3.3Mechanical forces experienced by cells can lead to nuclear deformation and flattening, and mechanosensing pathways are regulated by active and passive transport of proteins through nuclear pores At 1 h and 4 h there was no difference in cell density between tips and bases, but from 24 h onwards cells were significantly more crowded on the bases F. Quanti3.4The differences in the shape and motility of cells on tips and base of the topographies suggested there would be corresponding differences in the cytoskeleton and intercellular junctions. At 24 h we observed higher F-actin levels in cells on the tips than on the bases, correlating with the location of β1 integrin bright cells A and E. 3.5Since physical forces are transmitted between epithelial cells via intercellular junctions Quantification of Young’s modulus revealed that the stiffness of cells on the tips (3.9 ± 5.2 kPa) was significantly lower than that of cells on the base (18.0 ± 12.3 kPa) D. The stCell shape and ROCK regulate differentiation of epidermal stem cells 3.6To examine the influence of cell-cell junctions on biomechanical properties, keratinocytes were cultured on collagen-coated glass in low calcium FAD medium for 48 h to impair the formation of E-cadherin mediated adherens junctions. Following AFM measurements, the medium was changed standard  FAD. In a separate set of samples, ROCKi was added to standard FAD. Cells were incubated overnight and the AFM measurements were repeated. High levels of nuclear YAP and low levels of membrane E-cadherin expression were observed in low calcium FAD. After increasing the calcium concentration, YAP expression localised in the cytoplasm and E-cadherin expression increased significantly A.Fig. 5BThe AFM measurements indicated that the cell body of keratinocytes cultured in low calcium FAD displayed a lower Young’s modulus (2.0 ± 1.6 kPa) than keratinocytes cultured in standard medium. Switching cells from low calcium to standard medium led to a significant increase in cell body stiffness (4.1 ± 3.4 kPa), which could be prevented by ROCKi (1.4 ± 0.7 kPa) C. Under 3.7We hypothesised that stem cell patterning is controlled by forces exerted by cells on the slopes of the topographical features, rather than the tips or bases. To evaluate this, two additional topographies 4When primary human keratinocytes are seeded on a collagen-coated undulating polydimethylsiloxane (PDMS) elastomer substrate that mimics the topography of the epidermal-dermal junction in healthy, young skin, the cells become patterned such that the β1 integrin bright stem cells localise to the tips of the features While the dimensions of the PDMS substrates precluded comparing forces at intercellular junctions, we were able to measure them on flat substrates. We observed that intercellular junctions were stiffer than cell bodies and that their stiffness depended on Rho kinase. In future it would be interesting to discover whether forces exerted via intercellular junctions differ according to the slope of the undulations.We have previously reported that tip cells have nuclear YAP, a localisation that is dependent on Rho kinase and intercellular adhesion We have recently created a dynamic rig and shown that topographical features can impose patterning on a flat sheet of cells within 48 h Crowding in the epidermal basal layer, which we observed on the base of the topographies, is known to affect cell shape and can trigger movement into the suprabasal layer through a decrease in cortical tension and increased cell-cell adhesion Our finding that topography affects cell stiffness has implications for the changes in epidermal-dermal topography that occur in human skin with age and inflammation. The undulations decrease with age


As phosphorus (P) limits primary production in most lakes, protective and remediation efforts often seek to reduce P input. However, lower P concentrations do not always lead to lower O2 consumption rates. This study used a large hydrochemical dataset to show that hypolimnetic O2 consumption rates in seasonally stratified European lakes remain consistently elevated within a narrow range (1.06 ± 0.08 g O2 m−2 d−1) as long as areal P supply (APS) exceeded 0.54 ± 0.06 g P m−2 during the productive season. APS consists of the sum of total P present in the productive top 15 m of the water column after winter mixing plus the load of total dissolved P imported during the stratified season, normalized to the lake area. Only when APS sank below this threshold, the areal hypolimnetic mineralization rate (AHM) decreased in proportion to APS. Sediment trap material showed increasing carbon:phosphorus (C:P) ratios in settling particulate matter when APS declined. This suggests that a decreasing P load results in lower P concentration but not necessarily in lower AHM rates because the phytoplankton community is able to maintain maximum biomass production by counteracting the decreasing P supply by a more efficient P utilization. In other words, in-lake organic matter production depends only on APS if the latter falls below the threshold of 0.54 g P m−2 and correspondingly, the atomic C:P ratio of the settling material exceeds ~155.Areal oxygen (O This relation implies that primary production and hence phytoplankton density increases with P supply until maximum plankton density is constrained mostly due to self-shading in the water column4. Above this point, additional P no longer enhances organic matter (OM) production but instead results in excessive P accumulation within OM. This in turn leads to lower C:P ratios in the suspended and settling particles9.Since the 1970s, consensus on lake biogeochemistry has held that after light levels, P input exerts the strongest influence on primary production and by extension, trophic state2 depletion in deep waters where resulting anoxic conditions can potentially endanger aquatic life12. Mitigation strategies therefore typically focus on i) restriction of external P input, ii) in-lake P reduction13, and iii) aeration of O2-depleted water layers16. Research has shown that lowering external P loads results in lower P concentrations17 but not necessarily in lower hypolimnetic O2 consumption rates18.Excessive P input to lakes, reservoirs, and coastal marine ecosystems is thus interpreted to stimulate phytoplankton growth in their surface layer and subsequent O2 consumption24. Primary O2 sinks are (i) respiration and mineralization of OM settling through the water column25, (ii) the sediment surface, where settled OM is deposited and further processed, although at a potentially slower rate18, and iii) the lowermost water column layers, where oxidation of reduced sediment-borne compounds  released from the sediment are oxidized26. Consideration of the latter allowed consistent estimates of the O2 budget for areal hypolimnetic mineralization (AHM) rates27. As a consequence, the concentration of O2 limits AHM in shallow lakes while the deposition of OM determines AHM in deep lakes. Sedimentary OM deposition during a former hypertrophic period, as experienced by almost all anthropogenically-influenced lakes in the 1970s, will delay recovery of the lake’s AHM rate28.Almost a century of research has addressed hypolimnetic O2 consumption is conceptually well understood, the direct link between the annual P input and AHM is not as clear. In this study, we report the relationship between observed AHM rates and P supply from 21 European lakes of varying depth. The C:P ratios measured from settling material collected in sediment traps help to refine and better understand the elaborated relationship.While the relationship between settling OM and hypolimnetic O2 consumption. The areal consumption of O2 in the hypolimnion (AHM) is caused by (i) mineralization of OM settling from the productive zone, (ii) O2 diffusion into the sediment, and (iii) reduced compounds diffusing from the sediment. The contribution of nitrate to the mineralization of OM is only ~3% of the O2 consumption rate29. The structure of the food web may also exert influence on O2 depletion30 but - due to missing data - was not considered here.Biomass production in the epilimnion of a lake is limited by the total areal P supply (APS), i.e., the sum of total P present in the epilimnion at spring overturn plus the external supply of P available to phytoplankton  during summer stratification (see methods section). Phosphorus input during the productive season can be significant and may even exceed the P amount already present in the epilimnion after spring overturn. This latter parameter is often incorrectly used as the key reference for P available for production, neglecting that especially in oligotrophic lakes or lakes with a short water residence time, both P present in the trophic layer and seasonal P input contribute substantially to primary production and subsequent hypolimnetic O2 m−2 d−1 if APS exceeds 0.54 ± 0.06 g P m−2 , Sempach , and Hallwil  all of which are aerated with pure O2 consumption necessary for mineralization of organically bound carbon occurs at an observed ratio of 106:138 according to Eq. 131.The O2 consumption rate of 1.06 g m−2 d−1 shown in Fig. −2 over a stratified production period of six months. This value coincides with reported ranges of gross sedimentation from sediment traps and net sedimentation18. Only when the APS falls below the threshold of ~0.54 g P m−2 does the AHM rate decrease steeply in proportion to the P load. The transition of AHM from proportional increase with APS to the plateau is consistent with observations showing that net sedimentation of OM remains approximately constant when a threshold of P concentration is exceeded32. This suggests that phytoplankton can maintain maximum production under declining APS by incorporating less P relative to C up to a threshold of ~0.54 g P m−2.Varying proportions of P and N do not significantly influence this ratio. Accordingly, an O34 suggests that in a P-limited system OM production or mineralization are strictly proportional to P uptake or release, respectively. Accordingly, the blue dashed line in Fig. 2 consumption (AHM) relative to APS . Empirical data instead show that AHM decreased only if the available APS decreased below a threshold value of ~0.54 g P m−2 per productive season. The teal sector in Fig. 1 who stated that: “a body of water is in danger with regard to its trophic level when its springtime concentration of assimilable phosphorus compounds exceeds 10 mg P m−3and/or when the specific supply loading per unit area of lake reaches 0.2–0.5 g P m2−per year.”The Redfield stoichiometry Eq. 33,34 sug38. In contrast, under conditions of increasing P availability, plankton may take up P beyond its needs and store it, for example, as polyphosphates39. Combining the results presented in Figs. −2 or ii) the C:P ratio exceeds ~155. In lakes with a high APS the C:P ratio of settling material reaches 80 ± 10 (see supplementary note 1). This value ranges clearly below the classical Redfield ratio of 106 and supports the idea of excess P storage by phytoplankton. However, ‘luxury storage’40 does not continuously increase with the availability of P but appears to stabilize at a maximum level. According to Fig. −2. Consequently, results presented in Fig. Phytoplankton communities can apparently optimize carbon assimilation in case of diminished P availability during reoligotrophication. This manifests as increasing C:P ratios. The observed composition of settling particles collected by sediment traps in eight lakes – spanning a range of trophic states over 1- to 9-year observation periods - support the scenario described above. The C:P ratio of settling material increases with decreasing APS Fig.  and confhypo of less than 40 m that experienced an episode of eutrophic conditions with associated low hypolimnetic O2 concentrations in the past, accumulated large amounts of only partially degraded OM in the sediment18. The slow mineralization that occurs under anoxic conditions28 results in the formation of reduced compounds, e.g. methane and ammonium, which diffuse from the sediment to the water column26. Such fluxes of reduced compounds delay recovery from previously elevated AHM rates even once primary production has declined as a consequence of a lower APS. We consider 40 m as a critical hypolimnion depth because the O2 reservoir of such a lake is only about half way depleted by the end of the stratified season even if AHM rates reach maximum values (see supplementary note 2). In deep lakes (zhypo > 40 m), this effect subsides due to their large hypolimnetic O2 reservoirs and extended settling distances, permitting nearly complete aerobic oxidation of the OM already while settling through the water column25. To sum up, in deep lakes only minor amounts of OM accumulate in the anoxic sediment and the consequently low benthic fluxes of reduced substances cannot significantly delay their recovery after reduction of APS below the threshold of 0.54 g P m−2.Lakes with a mean hypolimnion depth z2 cycles. Data interpreted here reveal a distinct inflection point in the relationship between the areal hypolimnetic O2 consumption rate (AHM) and APS. Below an APS threshold of 0.54 g P m−2 per productive season, APS and AHM are proportionally related. Above this APS threshold, AHM rates take a constant value of 1.06 ± 0.08 g O2 m−2 d−1 independent of APS. Composition of settling particles collected by sediment traps affirms that the atomic C:P ratio of freshwater phytoplankton is highly variable and decreases strongly with increasing APS. Ratios of >155 indicate P limitation, whereas ratios <155 are characteristically observed in lakes that are excessively supplied with P. Thus, effective restoration of a lake suffering from too low hypolimnetic O2 concentration requires lowering its APS distinctly below the threshold value of 0.54 g P m−2. This refined understanding allows more accurate prediction of the minimum average hypolimnetic O2 concentrations observed at the end of summer stratification depending on a lake’s P supply, its mean hypolimnion depth and the hypolimnetic O2 concentration achieved by the end of the spring overturn ([O2]0).Water management agencies invest significant resources to lower P loads into natural water bodies. These expenditures are ultimately based on nutrient models seeking to minimize impact of the P load on the lakes’ Otol) preventing the hypolimnetic O2 concentration from decreasing below a desired minimum, [O2]min, by the end of the stratified period. As shown in Eq. tol depends on the O2 concentration attained by the end of spring overturn, [O2]0, the tolerable minimum average hypolimnetic O2 concentration at the end of the stagnation period, [O2]min, the mean hypolimnion depth, zhypo, and the duration of the stratified period Δtstrat.The effect of APS on AHM as depicted in Fig. 2]0 to attain 10 g m−3 and the desired [O2]min = 4 g m−3  and Δtstrat to extend over 180 days yieldsAssuming  is the TP concentration of the lake after spring overturn in [g m−3]. LP is the annual load of total dissolved P in [g yr−1], Vepi is the volume of the epilimnion in [m3], QApr-Sep and Q represent the water inflow for April to September and throughout the entire year in [m3], respectively, and zepi the epilimnion depth of 15 m. Epilimnion depth as well as water inflow values derive from averages over the past ten years. Water discharge data were available from the Hydrological Service of the Swiss Federal Office for the Environment (FOEN)42 and the Office of Waste, Water, Energy, and Air of the Canton Zürich (AWEL)43.[TP33 which results in:L is the lake volume [m3], and σ, β, and τ are the net sedimentation rate [yr−1], stratification factor [−], and hydraulic residence time [yr], respectively. Stratification factor β defined as the annual average ratio [TPepi]/[TPmix] was obtained from monitoring data. Net sedimentation rate σ was available in the form of direct sediment trap analyses from Lakes Sempach, Baldegg, Hallwil, and Sarnen or was estimated as 15 m/zL32, where zL is the mean lake depth. For lakes that showed a distinct trend in their TP content over the considered time period  in an autoclave. Elemental ratios were averaged from samples collected between April and September of the years indicated in Fig. Sinking particles were collected either with cylindrical plexiglass tubes measuring 65 cm in length and 9.2 cm in diameter or using automated sequential trapsSupplementary Information


Isotopic signatures (−60.2‰ to −7.6‰ for δ13Cch4 and −324.3‰ to +161.1‰ for Dch4), support a biogenic signature within waters emerging from the subglacial environment. Temperate glacial methane production and release may thus be a significant and hitherto unresolved contributor of a potent greenhouse gas to the atmosphere.The base of glaciers and ice sheets provide environments suitable for the production of methane. High pressure conditions beneath the impermeable ‘cap’ of overlying ice promote entrapment of methane reserves that can be released to the atmosphere during ice thinning and meltwater evacuation. However, contemporary glaciers and ice sheets are rarely accounted for as methane contributors through field measurements. Here, we present direct field-based evidence of methane production and release from beneath the Icelandic glacier Sólheimajökull, where geothermal activity creates sub-oxic conditions suited to methane production and preservation along the meltwater flow path. Methane production at the glacier bed (48 tonnes per day, or 39 mM CH The source of carbon essential for fuelling microbiologically-mediated reactions can be generated either in-situ at the bed of the glacier through chemolithoautotrophic production4, through utilising organic containing fossil soils7, or sourced from the surface environment8. Geogenic subglacial methane comprises abiogenic sources from subglacial volcanism, geothermal activity, and thermogenic sources through the thermal degradation of organic matter and subsequent migration of methane from hydrocarbon reserves to a stable sub-ice storage location e.g.9. Methane present at the bed of glaciers and ice sheets can then be retained in situ by high overburden pressures and low temperatures, which promote the entrapment of gases, or the storage of methane in hydrate reserves. Vast methane reservoirs potentially containing hundreds of petagrams of carbon could therefore accumulate and, if released as the ice melts, contribute positive feedback to rising atmospheric greenhouse gas concentration and global temperature3.The subglacial environment provides conditions suitable for the production and storage of methane. The presence of liquid water beneath temperate and polythermal ice masses, sub-oxic conditions due to poor hydrological connectivity, and carbon within basal sediments allow the survival of microbiological communities with the potential to produce biogenic methane eg.10 and favourable pressure–temperature relationships3 for methane production and storage in the subglacial realm, methane in glacial meltwaters has only been directly detected as aqueous methane in one study 11, or else inferred indirectly using δ13C of dissolved organic carbon12. For methane to be detected in meltwater outflow, sub-oxic conditions must occur not only at the glacier bed, but also throughout the subglacial drainage path. These conditions typically arise at the onset of subglacial discharge, during winter baseflow or as brief reversals in redox status of subglacial waters18. The inherently transient nature of these conditions thereby provides limited opportunity for direct field-based characterisation of methane production. Here, we examine an Icelandic glacier, Sólheimajökull, an outlet glacier of the Mýrdalsjökull icecap  and laboratory incubation of associated subglacial sediments demonstrate a strong potential for methanogenesis  this equates to a subglacial production capacity of 39 mM CH4 m−2 day−1. Using an upstream – downstream mass balance along the 4 km length (20 m width) of proglacial meltwater channel . This mass balance approach to calculating an evasive methane flux along the stream assumes minimal dilution, and no in-stream methanotrophy. Both are valid assumptions given the minimal input of additional meltwater between upstream and downstream sampling points, the minimal production/consumption of methane in the proglacial sediments . Evasion to the atmosphere was calculated as 54% along the 4 km stream reach, equating to 0.25 M CH4 m−2 day−1. Methane evasion from the Sólheimajökull sub-aerial stream network greatly exceeds mean flux values between river to atmosphere reported in the literature (4.23+\−8.41 mM CH4 m−2 day−1)22 indicating the potential significance of the subglacial methane source, if similar processes are also occurring at other glaciers.The appearance of elevated aqueous methane concentrations that are commensurate with the location and onset of subglacial drainage, suggests the environment of methane production must be beneath the glacier. Fluctuating discharge and changing subglacial methane concentrations on a seasonal basis preclude straightforward calculation of an annual methane flux from beneath the glacier. However, a typical summer season discharge of 50 mts Table , and the13Cch4 and Dch4. Isotopic fractionation during biogenic methanogenesis typically leads to δ13C values between −50 to −110‰, and δD values between −170 to −531‰23. Geogenic methane produced at high geothermal temperatures undergoes exchange with the surrounding water and mantle carbon, producing deuterium and carbon contents enriched in 2H and 13C respectively24. Signatures of mixed geogenic/microbial origin should therefore lie on an end member mixing trajectory as depicted in Fig. 13C and deuterium. As the most enriched values exceed the geogenic range, the observed isotopic signatures cannot be explained by a mixture of biogenic and geogenic methane ) and composition of residual methane (CH4(t)) is quantified following25 as α = 1.019 for 13C/12C, and for fractionation of D/H as α = 1.197. These incubation determined values of C and H enrichment during methanotrophy are encompassed within the published range of experimental values23, and result in relative changes to isotopic signatures during reaction progress that lie on a similar gradient to field data from this study . Under these conditions of distributed drainage (the ‘closed’ system), connectivity to the atmosphere is poor and dissolved gases can be depleted to produce meltwaters of low redox status. During the summer season, a discrete well-connected subglacial drainage system, characterised by well-defined conduits, expands up-glacier dependent upon the flux of surface run-off to the glacier bed, and typically follows the supraglacial snowline. Within this ‘open’ configuration, oxygen saturated meltwaters can drain rapidly from the surface of the glacier and through the subglacial system28. At polar glaciers of a polythermal nature, the drainage system displays similar characteristics, albeit with the winter slow flow component of the drainage system remaining sealed beneath the glacier until basal water pressures force a pressurised outflow, either shortly after the onset of the summer season29, or intermittently throughout the winter to produce characteristic proglacial icings e.g.30. However, at Sólheimajökull, the presence of the Katla geothermal area beneath the head of the glacier imparts profoundly different characteristics to meltwater discharge , together with methane produced during the summer season. During the winter season  of meltwater discharge. Greater headward expansion of drainage systems towards geothermal areas currently isolated beneath ice mass overburden would also ensure transport of meltwater and associated methane content to a position of sub-aerial degassing into the atmosphere. Pressure driven sub-oxia would likely become more prevalent with ice thinning until overwhelmed by the flux of oxygenated surface melt reaching the glacier bed or until ice disappearance. In this manner, this poorly quantified flux of sub-ice cap methane is likely an indirect, albeit self-reinforcing consequence of climatic change.The geothermally-influenced nature of the Sólheimajökull system is unusual in its ability to present a low redox status window which inhibits methane oxidation and preserves aqueous methane until the point of release from beneath the glacier. The dominance of biogenic methane production beneath an Icelandic temperate ice mass nonetheless raises the distinct possibility that methane generation could be proceeding undetected in other subglacial environments where the cocktail of temperate ice, low oxygen concentration, organic carbon and methanogenic communities coincide to promote methanogenesis. Increasing evidence for zones of strong geothermal activity beneath the West Antarctic Ice Sheet suggests that subglacial microbial communities with methanogenic potential may be more significant and extensive than previously anticipatedField samples were collected over restricted periods within the melt seasons of 2013 to 2017. Precise collection intervals are depicted in Table Samples for the determination of aqueous methane concentration were collected as a known volume of unfiltered water and stored within an airtight chamber with headspace at atmospheric pressure. Samples were left for 24 hours to undergo headspace equilibration and gases were then extracted through a sampling port and injected into evacuated exetainers  for later analysis of methane concentration and isotopic determination. Exetainers were over-pressurised to prevent ingress of atmospheric air and stored at ambient temperature to prevent vessel contraction and leakage. Headspace gas extraction at time t = 0 was used to determine background concentration prior to sample equilibration.4 m−2 day−1 following36.Proglacial sediments were monitored for the production and consumption of methane (methanogenesis and methanotrophy respectively) using static chambers (15 cm diameter × 10 cm height). Chambers were inserted into the sediments in triplicate at each site, and covered in aluminium foil to minimise temperature changes during the sampling period. Headspace gases were removed at set time intervals over a 45 minute incubation period to monitor the rate of methane production/consumption. Headspace gases were injected into evacuated exetainers which were over-pressurised to prevent the ingress of atmospheric air and stored at ambient temperature prior to further analysis for methane concentration. Flux values were calculated as µM CH37 were deemed the closest analogue to typical subglacial sedimentary deposits from the Sólheimajökull catchment. Sediments were incubated to determine the potential for methane production (methanogenesis) and consumption (methanotrophy) using standard procedures38. For each incubation type 10 grams of fresh weight sediment was added to a 100 ml sterilised Wheaton bottle and slurried with 20 ml deionised water. For anaerobic methane production incubations, the headspace was flushed with nitrogen gas to eliminate oxygen. For aerobic incubations the headspace was flushed with synthetic air, following which the methane concentration was adjusted to 150 ppm methane to assay for methanotrophy. Each set of incubations operated alongside control chambers supporting identical headspace conditions, but without the inclusion of sediment. All incubations were undertaken at a set temperature of 15 °C, reflecting optimal conditions for methane production and consumption, as established through preliminary testing. For methane production and oxidation potentials, triplicate samples were incubated for 49 and 7 days, respectively with regular sampling intervals during the periods of incubation . Fractionation between starting methane isotopic composition (CH4(i)) and composition of residual methane (CH4(t)) in the closed headspace is calculated following25:f is the fraction of methane remaining and δX is the isotopic composition of methane.Sediments extruded onto the glacier surface via thrust planes or melt out of fracture fill deposits c.f.4)  were analysed in every analytical sequence to encompass the expected sample concentrations. Standards were repeated at regular intervals39 to check for drift and ensure accuracy to within 95% of the true value. Sample triplicates had a precision (1S.D) representing <0.2% of the average value. The concentration of methane in water (Caq) is related to the concentration of gas measured in the headspace (Cg) via the dimensionless Henry’s Law solubility Constant (HCC) at a temperature of 273 K (0 °C)40.Methane concentrations were analysed using a PerkinElmer Autosystem XL Gas Chromatograph (GC)  fitted with a Flame Ionisation Detector (FID) operating at 300 °C. The GC was fitted with a stainless steel Porapak Q 50–80 mesh column  maintained at 60 °C. Three calibration gas standards  at the NERC Life Sciences Mass Spectrometer Facility, CEH Lancaster, UK. Manual injection volumes were dependent upon methane concentration and did not exceed 10 ml. Samples were initially passed through a Magnesium perchlorate/Carbosorb scrubber trap at 20 ml/min to eliminate water and CO2. Methane is oxidised in a combustion furnace using a braided platinum/copper/nichrome furnace wire inside a ceramic furnace tube of 200 mm × 0.4 mm i.d. heated in a furnace at 960 °C41. A preparation flow rate of 10 psi was required to give a flow rate of 20 ml/min through the furnace at full operating temperature. For δDch4, gas samples were purged from vials using a dual core needle and Helium carrier gas into a ThermoScientific precon concentration unit interfaced to a ThermoScientific Delta V plus isotope ratio mass spectrometer at UC Davies, University of California, USA. Cryogenic trapping and GC separation followed by pyrolysis at 1450 °C yielded H2 for determination of D/H ratios of methane gas42. δ13C values were corrected to VPDB using working CH4 standards cross calibrated with a CO2 reference gas, calibrated to NIST REF-Heavy Palaeomarine Origin (CO2) (RM 8562) and NIST REF-Biogenic Modern Biomass Origin (CO2) (RM 8564). The reproducibility of δ13Cch4 was better than ±0.2‰. δD was corrected to VSMOW using reference gasses calibrated to international standards NIST 8559, 8560, and 8561. Within-run standard replication of both samples and standards (1SD) was better than 2.6‰ for Hydrogen.Supplementary information