The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild‐type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity  in GM1‐enriched domains only, thus establishing a link between aggregate‐membrane contact and cell damage.The first genetic variant of β The protein acts as a chaperone during the assembly of MHC I quaternary structure, an event that is required for antigen presentation 2+ ions βin vitro under physiological conditions, many recombinant b2M variants have been investigated, and hypotheses about the mechanisms underlying protein fibrillogenesis have been put forward T state, which, albeit being globally folded, bears the His31‐Pro32 peptide bond in the non‐native trans configuration cis‐trans isomerization in vitro under conditions where the stability of the wild‐type protein is unaffected, prompting a re‐evaluation of previously hypothesized mechanisms of b2M fibrillogenesis. Individuals of this family suffered a complex set of clinical symptoms, including bowel dysfunction with chronic diarrhoea and sicca syndrome, weight loss and postural dizziness 123I‐human serum amyloid protein (SAP) scans and postmortem examination of affected people revealed the presence of amyloid deposits in the spleen, liver, heart, peripheral nerves, salivary and adrenal glands. Importantly, the D76N b2M present in amyloid fibrils extracted from patients consisted only of the full‐length variant without any truncated form. Moreover, in contrast to DRA patients, all the members of this family displayed normal circulating concentrations of b2M and normal renal function. Consequently, the pathological condition of these patients was described as a hereditary systemic amyloidosis associated with D76N b2M Although conversion of wild‐type b2M into amyloid fibrils is difficult to achieve in vitro at a protein concentrations 20‐fold higher than those found in patients under dialysis, wild‐type b2M remains predominantly monomeric for several months T state is significantly more populated in the variant. Indeed, the in vitro equilibrium concentration of such conformation, measured at physiological pH and in the absence of denaturants, appears increased in the case of the variant, with values of 4.8 ± 3.0% and 25 ± 9.0% for wild‐type and D76N b2M, respectively The experimental evidence described above points towards a high amyloidogenic potential of D76N b2M. In fact, when incubated in vitro stability and aggregation propensity of D76N b2M, additional information is still required to link these data to amyloid‐related toxicity of this variant. In this article, we exploited a battery of complementary biophysical tools to characterize the species transiently populated during D76N aggregation. The ability of such aggregates to interact with model membranes bearing different lipid composition was also evaluated. The outcome of these experiments was compared with cell biology studies aimed at investigating the mechanisms of cytotoxicity of the different aggregation states of D76N b2M. These results provide an unprecedented characterization of the pathway spanning from protein misfolding to the cell dysfunctions underlying b2M hereditary systemic amyloidosis.Although the pieces of evidence described above provide valuable information concerning the D76N b2M was expressed and purified as previously reported °C in PBS. The spectrum was collected over the 190–250 nm wavelength range using a Jasco‐810 Spectropolarimeter equipped with a thermostated cell holder attached to a Thermo Haake C25P water bath . A 1.0‐mm path‐length cell was used. The spectrum was blank subtracted and converted to mean residue ellipticity per residue (θ).A far‐UV circular dichroism (CD) spectrum of 0.2 mg/ml D76N b2M was acquired at 25Rhfolded = 4.92N0.285, where N is the number of residues and Rhfolded is the hydrodynamic radius expressed in Å Size distribution analysis was carried out at 25°C using a Malvern Zetasizer Nano S dynamic light scattering (DLS) device  on 20 μM D76N b2M samples. Each sample was analysed considering the refraction index and viscosity of its dispersant. A 10‐mm reduced volume plastic cell was used. The acquired data were compared with the theoretical hydrodynamic radius expected for folded polypeptide chains, calculated as i) 60 μl of the aggregating protein solution and 440 μl of a solution containing 20 μM CR (spectrum 1), (ii) 60 μl of PBS devoid of protein and 440 μl of buffer containing 20 μM CR (spectrum 2) and (iii) 60 μl of protein and 440 μl of PBS in the absence of CR (spectrum 3). The spectra were recorded from 400 to 700 nm using a Jasco V‐630 spectrophotometer and a 5.0‐mm reduced volume quartz cell. The difference spectra were obtained by subtracting spectrum 2 and spectrum 3 from spectrum 1.Congo Red  spectra were recorded at 25°C in the presence of D76N b2M. For any time‐point, three spectra were acquired containing  Aliquot of 62 μl of the aggregation solution was mixed with 438 μl of a solution containing 25 μM thioflavin T ThT dissolved in a 25 mM phosphate buffer, pH 6.0. Fluorescence spectra from 450 to 600 nm were acquired at 25°C in a 2 × 10 mm quartz cuvette using a Perkin‐Elmer LS 55 spectrofluorimeter  equipped with a thermostated cell holder attached to a Haake F8 water bath. Excitation wavelength was 440 nm. The ThT fluorescence spectrum obtained under the same conditions without D76N b2M was subtracted from that acquired in the presence of the dye. The data were fitted to an empirical sigmoid function Samples containing aggregating D76N b2M at 20 μM were investigated for their ability to bind 8‐anilinonaphthalene‐1‐sulfonic acid . Briefly, 100 μl of the aggregation solution were mixed with 500 μl of a solution containing 120 μM ANS in PBS. Then, the fluorescence spectra were recorded in the 400–600 nm range using the same equipment and cuvette described above for the ThT fluorescence experiments. Excitation wavelength was 370 nm. The data were analysed by subtracting blank spectra consisting of the dye in PBS without protein.Transmission electron microscope (TEM) imaging was carried out as follows. Briefly, 5.0‐μl aliquots of D76N b2M were withdrawn at different aggregation times, loaded onto a formvar/carbon‐coated 400 mesh nickel grids  and negatively stained with 2.0% (w/v) uranyl acetate (Sigma‐Aldrich). The grid was air‐dried and examined using a JEM 1010 TEM at 80‐kV excitation voltage.2 incubator. All the materials used for cell culture were from Sigma. HL‐1 mouse atrial myocytes were obtained from Dr W. C. Claycomb  and grown in T25, gelatin/fibronectin‐coated flasks, as previously described N,N‐dimethylformamide, pH 4.7) was added to each well and the samples were incubated at 37°C to allow complete lysis. The absorbance values of blue formazan were determined at 595 nm with an automatic plate reader . The final absorption values were calculated by averaging three independent measurements of each sample and subtracting from this the average of the blank solution, consisting of 100 μl of MTT solution and 100 μl of lysis buffer . All data were expressed as mean ± S.D.The SH‐SY5Y cells were cultured at 37°C in complete medium , in a humidified, 5.0% CO2 DCFDA; Sigma‐Aldrich). CM‐H2 DCFDA is a cell‐permeant indicator for ROS that is not fluorescent until removal of the acetate groups by intracellular esterases and subsequent oxidation. The latter can be detected by monitoring the increase in fluorescence at 538 nm. The SH‐SY5Y and HL‐1 cells were plated at a density of 10,000 cells per well on 96‐well plates. After 24 hrs of cell exposure to the aggregates, 10 μM DCFDA in DMEM without phenol red was added. After 30 min, the fluorescence values at 538 nm were detected by Fluoroscan Ascent FL .The intracellular levels of reactive oxygen species (ROS) were determined using the fluorescent probe 2′,7′–dichlorofluorescin diacetate, acetyl ester . Subconfluent SH‐SY5Y cells cultured on glass coverslips were incubated at 37°C for 5.0 min with 5.0 μM Fluo‐3 AM prior to exposure to D76N b2M aggregates for 1.0 hr. At the end of the incubation, the cells were fixed in 2.0% buffered paraformaldehyde for 10 min. Cell fluorescence was visualized using a confocal Leica TCS SP5 scanning microscope  equipped with a HeNe/Ar laser source for fluorescence measurements. The observations were performed using a Leica Plan 7 Apo X63 oil immersion objective, suited with optics for DIC acquisition. Cells from five independent experiments and three areas (about 20 cells/area) per experiment were analysed. The fluorescence intensity of Fluo3‐AM was analysed by using ImageJ software  and expressed as arbitrary units.The cytosolic levels of free CaCell apoptosis was detected by the Annexin V‐fluorescein isothiocyanate (FITC) Apoptosis detection kit (Sigma‐Aldrich). A propidium iodide (PI) solution containing Annexin V‐FITC was used to discriminate viable from apoptotic and secondary necrotic cells. Briefly, after a 24‐hr treatment with the most toxic D76N b2M aggregates, the cells were incubated with Annexim V‐FITC and PI for 10 min, at room temperature. Then, the cells were analysed by flow cytometry. Annexin V‐FITC was detected as a green fluorescence at 514 nm and PI was detected as a red fluorescence at 617 nm.Subconfluent SH‐SY5Y and HL‐1 cells grown on glass coverslips were treated for 24 hrs with the different D76N b2M aggregates grown for various time lengths, at 5.0 μM final concentration. After incubation, the cells were washed with PBS; then, GM1 labelling at the cell surface was performed by incubating the cells with 10 ng/ml CTX‐B Alexa488 in complete medium for 10 min at room temperature. Finally, the cells were fixed in 2.0% buffered paraformaldehyde for 10 min., permeabilized by treatment with a solution containing 50% acetone and 50% ethanol for 4.0 min. at room temperature, washed with PBS and blocked with PBS containing 0.5% BSA and 0.2% gelatine. After blocking, the cells were incubated for 1.0 hr at room temperature with a rabbit polyclonal antibody raised against b2M  diluted 1:600 in the blocking solution and washed with PBS for 30 min. under stirring. The immunoreaction was revealed with Alexa 568‐conjugated anti‐rabbit secondary antibody (Invitrogen) diluted 1:100 in PBS. Finally, the cells were washed twice in PBS and once in water to remove non‐specifically bound antibodies. Cell fluorescence was visualized using a confocal Leica TCS SP5 scanning microscope (Leica) equipped with a HeNe/Ar laser source for fluorescence measurements. The observations were performed using a Leica Plan 7 Apo X63 oil immersion objective, suited with optics for DIC acquisition.To reduce cell membrane GM1, the glucosylceramide synthase of the plated cells was inhibited by supplementing the cell culture medium with 25 μM D‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol  for 72 hrs at 37°C in complete medium as previously described Quantum dots (QDs) labelling and live imaging have been extensively described x(t), y(t)) by computing the MSD xi and yi are the coordinates of a particle on frame i, dt is the time between two successive frames, N is the total number of frames of the trajectory and ndt is the time interval over which the displacement is averaged. This function enables the analysis of the lateral dynamics on short‐  and long‐ (types of motion) time scales. Different types of motion can be distinguished from the time dependence of the MSD t) = 4D2‐5t + b. The cumulative probability C(d) of D defines the probability that D is less than d. We compared cumulative probability distributions and median instead of mean values because D values were spread over four orders of magnitude. The Images were thresholded with ImageJ software, creating binary masks corresponding to amyloid aggregates.The mean square displacement (MSD) analysis allows to calculate the initial diffusion coefficient (D) of each particle 2 solution were deposited on a 1.0 cm × 1.0 cm freshly cleaved mica substrate. In order to get uniform bilayer coverage, the samples were incubated for 15 min. at 60°C after 10 min. storage at room temperature; then the samples were kept at room temperature in a close chamber with 100% relative humidity. Before atomic force microscopy (AFM) measurements, SLBs were gently rinsed three times with Milli‐Q water to remove excess vesicles from the liquid subphase.1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine , sphingomyelin (SM)  and ganglioside GM1  were purchased from Avanti Polar Lipids ; cholesterol (Chol), chloroform and methanol were purchased from Sigma‐Aldrich. Two different mixtures were prepared, containing DOPC:SM at 2:1 molar ratio, a fixed, 1.0%, molar percentage of cholesterol and a variable percentage of 5.0% GM1, when present. To prepare the liposomes in solution, we dissolved the phospholipid powder in chloroform/methanol (2:1) according the desired composition and gently evaporated the mixture to dryness under a nitrogen flux. Aliquots were stored overnight under vacuum and resuspended in Milli‐Q water to form multilamellar vesicles. Multilamellar vesicles suspensions were pre‐sonicated at 60°C for 1.0 hr and then extruded 11 times through polycarbonate membrane with 100 nm pores using a commercial extruder (Avanti Polar Lipids) at 60°C. The suspensions were left to cool at room temperature and the large unilamellar vesicles suspensions so obtained were diluted 10‐fold in Milli‐Q water; then 40 μl of each suspension and 10 μl of 10 mM CaClin situ, using the thermal noise method. D76N b2M aggregates at different times of aggregation  were administered to the sample under the AFM head at a 5.0‐μM final concentration and let incubating for 20 min. before further measurements. Before injection, the AFM tip was lifted up 60 μm from the sample. Quantitative Imaging mode (QI–JPK Instruments) was used to determine sample topography and elasticity (Young's Modulus) before and after the administrations of the protein aggregates. QI is based on the acquisition of a large set of force–distance curves and on the reconstruction of sample topography from the tip position at the specific force load. Local mechanical properties of the sample can be extracted from the analysis of the acquired curves. QI images deriving from 128 × 128 force–distance (FD) curves were acquired on different samples before and after protein administration, with maximum force load of 1.0 nN. For each curve, the tip speed was 25 μm/sec. and the curve length was 100 nm. For absolute value determination of the Young's Modulus, each single FD curve was fitted with the Hertz model, the tip was approximated as spherical and with a radius of 30 nm. Comparisons between the different cumulative distributions were performed by the Kolmogorov–Smirnov test. A P‐value <0.05 was considered statistically significant. SPT data were collected from 10 cells from three independent experiments.AFM images and indentation measurements were performed by using a Nanowizard III  mounted on an Axio Observer D1 . inverted optical microscope. V‐shaped DNP silicon nitride cantilevers , with a nominal spring constant 0.06 N/m, resonance frequency in air in the 40–70 kHz range and tip typical curvature radius of 20–60 nm were used. The actual spring constant of each cantilever was determined in vitro aggregation of the protein was initiated at 37°C in PBS at a concentration of 20 μM under vigorous shaking. Aliquots of the mixture were withdrawn at different time intervals since the beginning of the aggregation reaction, and a number of biophysical properties of the aggregates found in the mixture were investigated  and CM‐H2DCFDA, respectively. Figure 2+ concentration in cells treated for 1.0 hr with 5.0 μM D76N b2M aggregates aged 72 or 144 hrs, respectively, whereas no significant changes were observed when the cells were treated for 1.0 hr with vehicle (PBS) or with protein samples obtained at early aggregation times (up to 24 hrs), in agreement with the MTT data. Since derangement of intracellular free Ca2+ levels and ROS production are biochemically related parameters, we also measured the amount of ROS levels in SH‐SY5Y cells treated with D76N b2M samples aggregated for different time periods. Figure 2+ and MTT results. The same results were obtained with HL‐1 cells  confirming that the 144‐hr‐aged aggregates were the most aggressive form.We also investigated whether the species transiently populated along the aggregation path of D76N b2M were toxic and, if so, whether toxicity was associated to membrane binding and/or aggregate internalization. The cytotoxicity in our cell model was studied using human SH‐SY5Y neuroblastoma cells previously shown to be sensitive to the toxicity of 20 μM b2M aggregates Finally, we investigated whether the aggregate‐exposed cells underwent death and whether the latter was of necrotic or apoptotic type, considering that apoptosis and necrosis can both be triggered by amyloid aggregates in vivoIt is widely reported that a main mechanism of amyloid aggregate cytotoxicity requires the primary interaction between the aggregates and the cell membrane, resulting in functional and/or structural perturbation of the latter. To investigate the ability of variously aged D76N b2M aggregates to interact with the cell membrane of the exposed cells, we performed confocal microscopy experiments using both a polyclonal antibody raised against recombinant b2M and Alexa 488‐conjugated CTX‐B, a probe specific for the monosialotetrahexosylganglioside 1 (GM1), a common lipid raft marker widely reported as a key interaction site for amyloids In another set of experiments, we sought to confirm that aggregate toxicity resulted from their ability to interact with the cell membrane at GM1‐enriched sites and hence was dependent on the presence of GM1 in the membrane itself. To this purpose, we modified the ganglioside content in the membrane by pre‐treating the cells with 25 μM PDMP, a glucosylceramide synthase inhibitor that blocks the natural synthesis of GM1 −1 μm2/sec. with respect to 2.9 × 10−2 μm2 sec. and 2.7 × 10−2 μm2/sec. This discrepancy is possibly due to the higher acquisition frame rate used here (100 Hz as compared with 3 Hz). Nevertheless, the absolute value of the change of GM1 D in the presence of D76N b2M aggregates was comparable to that observed in the presence of Aβ1‐42, amylin and Sup35pNM amyloid aggregates Single particle tracking experiments were performed to confirm the interaction between GM1 and D76N b2M aggregates aged 144 hrs at the plasma membrane of SH‐SY5Y cells. This technique exploits fluorescent semiconductor nanocrystals, QDs, as a very bright and photostable probe to follow the motion of membrane molecules such as lipids and proteins with nanometric resolution in living cells To confirm, and get further insights on the aggregate‐membrane interaction, we also investigated by AFM imaging and elasticity measurements whether aggregate binding affected the physical properties of synthetic lipid bilayers. D76N b2M aggregates were administered to synthetic lipid bilayers with two different lipid mixtures (DOPC:SM:chol:GM1 and DOPC:SM:chol), as described in Materials and Methods. The morphology and elasticity of the bilayers were assessed before and after addition of the aggregates. Both lipid mixtures assembled into a lipid bilayer with two distinct lipid phases. The thicker domains correspond to the ordered lipid phase (LO) that, under our conditions, was enriched in cholesterol, sphingomyelin and GM1, when present. The rest of the sample represents the disordered phase domain (LD), where the DOPC is the main component in vitro in PBS. The determination of the three‐dimensional structure and the mutation‐related behaviour of this variant have provided valuable information about the molecular basis of D76N b2M aggregation D76N b2M is the first described genetic variant of b2M associated with a familial form of systemic amyloidosis. The protein displays a stronger amyloidogenic propensity with respect to the wild‐type form and aggregates easily in vitro aggregation of many different proteins are the main culprit of amyloid‐related toxicity It has been long recognized that the unstable, heterogeneous pre‐fibrillar assemblies transiently populated during the early phases of It is likely that the interaction between aggregates obtained after 144 hrs of incubation and membrane surfaces results in membrane damage and cytotoxicity through a mechanism involving the release of cytotoxic species, as hypothesized elsewhere 2+ and ROS levels are amongst the earliest modifications associated with the membrane perturbations that underlie cell suffering in aggregate‐exposed cells, our data further support the establishment of GM1‐aggregate interaction as a key switch of amyloid cytotoxicity. Our measurements with AFM in QI mode on model membrane reconstructed on a mica surface further support previous results and, in addition, confirm GM1 interaction as an event required for cytotoxicity. Furthermore, these data point to possible toxicity mechanisms based on a perturbation of the elasticity (E) of specific (GM1 enriched) ordered lipid phases (LO) following aggregate interaction with the cell membrane, as observed for other amyloid‐related damaging systems The observations arising from our study raise the possibility that the toxicity of protein aggregates is not necessarily related to the oligomeric nature but rather to structural and surface properties that may or may not be shared by early and late aggregates. Our findings showed that differently from many other aggregating peptides and proteins, the mature aggregates of D76N b2M displayed the most severe cytotoxic effects, in agreement with the damage induced by wild‐type b2M fibrils In conclusion, our data provide a first glimpse on the aggregation path, membrane interaction and cytotoxicity of the only known b2M variant associated with a familial form of systemic b2M amyloidosis. Further investigations will define more precisely the molecular and environmental players affecting D76N b2M aggregation in tissues, the molecular details and the biochemical and cell biology modifications that underlie the physiological effects of those species. When added to those pertaining wild‐type b2M, these data will aid depicting more precisely the scenario of b2M deposition, providing clues for the search of molecules and treatments aimed at tackling the pathologies associated with this phenomenon.The authors confirm that there are no conflicts of interest.Figure S1 AFM topography and elasticity maps of lipid bilayers obtained from QI mode measurements. Morphology of lipid bilayers enriched in GM1 (5%) in presence of aggregates of 5.0 μM D76N b2M aged 1 hr (A) or 72 hrs (B). The white arrows show accumulated protein aggregates; the red ‘x’ marks the absence of LO phase. The morphology of lipid bilayers lacking GM1 was not altered upon interaction with protein aggregates aged 1.0 hr (C) or 72 hrs (D). Height distributions in the presence (I and J) or in the absence (K and L) of GM1. Elasticity maps in the presence (M and N) or in the absence (O and P) of GM1 and the corresponding Young's modulus. LO: ordered lipid phase; LD: disordered lipid phase; PA: peak corresponding to the signal arising upon accumulation of aggregates onto LO.Click here for additional data file.Figure S2 Cytotoxicity of D76N b2M aggregates on HL‐1 cells. (A and B) HL‐1 cells exposed for 24 hrs to 5.0 μM D76N b2M aggregated for 24 hrs (A) and 144 hrs (B). The cells were stained with Alexa 488‐conjugated CTX‐B (green fluorescence); protein aggregates were stained with anti‐b2M antibodies followed by treatment with Alexa 568‐conjugated anti‐rabbit secondary antibodies (red fluorescence). FRET efficiency is shown in panels 1, 2 for aggregates aged 24 hrs or 144 hrs, respectively. (C) MTT assay on HL‐1 cells exposed for 24 hrs to 5.0 μM D76N b2M samples aggregated for different times. (D) ROS production in HL‐1 cells exposed for 24 hrs to D76N samples (5.0 μM) aggregated for varying lengths of time. Error bars in all bar plots indicate the standard deviation of three independent experiments carried out in triplicate. T‐test analysis: *P < 0.005; **P < 0.001;***P < 0.0001 versus untreated cells.Click here for additional data file.


This study presents a novel procedure for accelerating methanogenesis from wastewater by adding elemental manganese into the anaerobic digestion system. The results indicated that elemental manganese effectively enhanced both the methane yield and the production rate. Compared to the control test without elemental manganese, the total methane yield and production rate with 4 g/L manganese addition increased 3.4-fold (from 0.89 ± 0.03 to 2.99 ± 0.37 M/gVSS within 120 h) and 4.4-fold (from 6.2 ± 0.1 to 27.2 ± 2.2 mM/gVSS/h), respectively. Besides, more acetate consumption and less propionate generation were observed during the methanogenesis with manganese. Further studies demonstrated that the elemental manganese served as electron donors for the methanogenesis from carbon dioxide, and the final proportion of methane in the total generated gas with 4 g/L manganese addition reached 96.9%, which was 2.1-fold than that of the control (46.6%). Finally, methanogenic archaea utilize the acetic acid and H2 as substrates to produce methane. Though acetate as methanogenic substrate, H2 is also an important precursor to hydrogenotrophic methanogens, which can use H2 as electron donors for biosynthesis of methane by reducing CO22 and CO2) coupled to hydrogenotrophic methanogenesis was a dominant pathway in thermophilic methanogenic reactors789Comparing with the aerobic treatment, biological methanogenesis from wastewater under anaerobic conditions is widely recognized as a highly sustainable waste treatment process due to the conversion of wastewater’s organic content into renewable bioenergy in the form of methane rather than waste activated sludge1242, the intermediate for methane synthesis, could be produced from organic wastes by biological fermentation, but yields were limited2 seemed to be an efficient method. Recently, bioelectrochemical systems were widely utilized to assist microbial production of methane and hydrogen from wastewater and waste activated sludge122, which obtained electrons from the cathode, to reduce the CO2, produced by acetate oxidation or graphite anode oxidation2 through cathodic depolarization1517et al. (2014) indicated that the addition of zero valent iron could enhance CH4 production rate by 43.5% in anaerobic digestion of waste activated sludgeH17et al. 14 indicaet al. previously demonstrated that methanogenesis by Methanobacterium thermoautotrophicun could occur coupled to the anaerobic oxidation of manganese and some other metals2 and be more effective than iron element due to its stronger reducibility.Manganese metal, a reductive material, offered many similar physicochemical properties compared to elemental iron. Lorowitz Therefore, the main aim of the present study was to explore a simple and efficient method for accelerating the biological methanogenesis through adding elemental manganese into an anaerobic digestion system. To our best knowledge, it was the first time to enhance this process by elemental manganese addition. In order to evaluate the methanogenesis performance, methane production rates with different dosage of elemental manganese addition were measured, volatile fatty acids and pH with and without elemental manganese addition were determined, and the effects mechanism of elemental manganese on biological methanogenesis was also investigated.During the batch experiment, glucose was utilized as organic carbon source, which could be fermented to VFAs rapidly. Therefore, the methanogenesis process was proved to be the rate-determining step2 concentrations were also detected during the whole research and the results were shown in 2 concentrations reached a peak in 12 hour and then it began to decrease mainly due to the beginning of methanogenesis process. As predicted, an obvious difference in the CO2 concentrations was observed with elemental manganese added. The consumption rates of CO2 with elemental manganese additions were much higher than that of the control. Moreover, after 46 hours, nearly no CO2 accumulations were determined with 4 g/L and 8 g/L elemental manganese added and a much lower accumulation was observed with 2 g/L elemental manganese added than that of the control, which showed a precipitate rise between 36 and 60 hours and then remained at a steady level. The difference of CO2 amount between the control and the 4 g/L elemental manganese assays was about 0.93 M/gVSS. Therefore, it was reasonable to consider that the additional CH4, generated with manganese added was converted from the CO2.The CO2 into CH4 with hydrogen as substrate4 formation from CO2 through cathodic depolarization15Electrons, generally provided by hydrogen, were needed to accomplish the conversion from CO2.These results indicated that the additional manganese could act as electron donors, which was similar with other metals4 production rates and manganese ions concentrations showed that the CH4 production rate was significantly increased in the presence of manganese ions  analysis was conducted using a FEI Company’s Quanta 450 field emission scanning electron microscope to verify the possible generated manganese oxides based on such hypothesis. Firmicutes, a kind of well-known manganese-oxidizing bacteria26The observation of manganese oxides indicated the existence of manganese-oxidizing process in the anaerobic digestion system. In order to investigate the functional strains during the manganese ion oxidation, clone library analysis of 16S rRNA genes of the seed inoculum (Bacteria) was conducted. The results indicated that These results showed that appropriate dosages of manganese addition could not only enhance the methanogenesis process, but also avoid the potential pollution that caused by manganous ions2, the precursor for hydrogenotrophic methanogenesis232 as an intermediate and further accelerating methanogenesis was reasonable.The pH value was an important influence factor for biological methanogenesis. The initial pH values of the anaerobic system were adjusted to 7.2 ± 0.1 with 0.1 M HCl and the final pH values were 5.5 ± 0.1 (control), 7.4 ± 0.1 (2 g/L), 8.4 ± 0.1 (4 g/L) and 8.9 ± 0.1 (8 g/L), respectively, presenting an obvious increase trend with higher elemental manganese levels. During the methanogenesis process, the pH values could increase due to the consumption of protons, which were utilized to form H4 was determined with CO2 consumption, it was not ascertained whether the conversion was mediated by microbiological process. For such reasons, two kinds of methods were utilized to verify whether the excess CH4 production process was catalyzed by microorganisms.Although excess CH4 was determined during the whole operation period, though the CO2 decreased to 6.59 ± 0.31 M at 46 h. Moreover, a much higher level of manganous ions of 415.0 mg/L was observed in the end. It was reasonably considered that the high CO2 pressure in the gas phase resulted in an increase in the bicarbonate ion in the liquid phase, which accelerated the cathodic depolarization of elemental manganese. Furthermore, the created manganous ions could not be further oxidized in the absence of the manganese-oxidizing microorganisms. Therefore, the manganous ions concentration in this test was higher than that of the batch experiments. Besides, due to the lack of methanogens in the anaerobic system, the consumed CO2 could not be utilized to synthetize CH4, though electrons could be provided by the elemental manganese. These results indicated that the simultaneous consumption of CO2 and manganese was mediated by chemical reaction and microorganisms were required to participate in the CH4 production process.First, the anaerobic sludge was removed from the digestion system and other conditions were kept the same as the batch experiment with 4 g/L manganese added. 2 in the presence of microorganisms, which might cause the increase in the CO2 contents after 46 h. The high manganous ions concentration obtained in this test could also imply the existence of manganese-oxidizing microorganisms in the anaerobic sludge.In addition, since the digestion medium was prepared without sterilization, the organic contents could be converted into COSecondly, one kind of methanogenesis inhibitor, 2-bromoethanesulfonate (BES) was added into the medium with complete digestion system containing sludge. 4 g/L manganese was supplied into the system and the test was carried out under the same conditions as mentioned above except for addition of 20 mM BES.4 and CO2 productions with BES added were shown in 2 was rapidly generated before 12 h by the acidogenesis and acetogenesis processes. A sudden decrease trend in CO2 was detected with manganese added, while it showed a slow increase trend in the absence of manganese. Though manganous ions were also determined with BES added  EDTA·Na2 15, ZnSO4·7H2O, CuSO4·5H2O 0.15, CoCl2·6H2O 0.24, MnCl2·4H2O 0.99, NaWO4·2H2O 0.05, NaMoO4·2H2O 0.22, NiCl2·6H2O 0.19, NaSeO4·10H2O 0.21 and H3BO4 0.014 was used throughout the study. Glucose and carbon dioxide were used as carbon sources according to different requirements. Elemental manganese power  was bought from Aladdin@.The anaerobic sludge utilized as initial inoculum was collected from a local domestic sludge treatment plant in Dalian. A synthetic wastewater consisting of (g/L) the following: peptone 0.1, yeast extract 0.01, NaHCOInvestigation of the effects of elemental manganese on methanogenesis was carried out through batch experiments with different manganese concentrations . The manganese powder was added into the anaerobic digestion system at the beginning with an aliquot (50 mL) of the synthetic medium, which contained 1 g wet weight sludge (MLVSS of 1.8 g/L) and a chemical oxygen demand (COD) of 1.0 g/L with glucose as carbon source. The fermentation system was purged with dinitrogen gas for 10 minutes to removal dissolved oxygen in the liquid phase and air in the head space. The initial pH of the medium was adjusted to 7.2 ± 0.1 with 0.1 M HCl and the temperature was maintained at 35 ± 1 °C in a water bath shaker at a shaking speed of 150 rpm to keep full contact between the sludge and the medium. The samples were collected regularly using a sterile syringe for the aqueous samples and a gastight syringe for the gaseous samples, respectively.2 was aerated into the system to investigate the potential conversion of CO2 into CH4 under such conditions. The experiments lasted for 118 hours and gaseous samples in the headspace were collected with a gastight syringe punctually for the determination of CH4. Subsequently, in the other set, 2-bromoethanesulfonate (BES), a kind of widely used methanogenesis inhibitor was added into the digestion system for further investigation. The digestion system contained 50 mL synthetic medium with 1.8 g/L MLVSS and 20 mM BES and glucose (1.0 gCOD/L) was used as carbon source during this test. 4 g/L manganese was added into the system with the intension of studying the function of manganese during the digestion process. Systems with no manganese addition was designed as control. The experiments lasted for 108 hours under the same conditions as mentioned above.Afterwards, in order to verify that the enhanced methane production with elemental manganese added was achieved by microbial catalysis, two sets of batch experiments were performed. In the first set of experiments, no anaerobic sludge was supplied into the digestion system and the system consisted of 50 ml synthetic medium and 4 g/L elemental manganese powder. Besides, a certain concentration of CO2 with elemental manganese added, test with CO2 as sole carbon source was conducted. The anaerobic digestion system contained 50 ml synthetic medium and 44.4 M CO2. 1 g wet weight sludge (MLVSS of 1.8 g/L) and 4 g /L elemental manganese were added into the system, respectively. The test lasted for 146 hours and was operated under the same conditions as mentioned above. All the data shown in the present study were the mean values of triplicate experiments.Finally, to study the feasibility of methanogenesis from CO−1. The oven was set at 100 °C and the injector and detector were maintained at 100 and 120 °C, respectively. Standard curves of each measured gas were established by injecting a known volume of high purity standard gas into the GC.Gases (methane and carbon dioxide) accumulated in the head space were periodically analyzed. 0.5 mL of gas was taken from the vial head space with a gastight syringe and directly injected into a gas chromatography  with a thermal conductivity detector (TCD) and a stainless steel column . Helium gas was used as the carrier gas with a flow rate of 30 mL minOxidized manganese, Mn (II) immediately produced in the aqueous phase, treated as a key factor investigating the oxidation of elemental manganese was quantified spectrophotometrically using the formaldoxime methodsClone libraries were constructed from samples (Bacteria) of the seed inoculum. Bacterial 16S rRNA genes were amplified with forward primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer 1392R (5′-ACGGGCGGTGTGTRC-3′)Triplicate PCR products were pooled and purified with Qiaquick PCR Gel Extraction Kit , cloned using the pMDTM18-T Vector System  with TOP10 competent Escherichia coli cells  and plated on LB (Luria-Bertani) plates supplied with ampicillin (Sigma). Colonies were randomly picked, cultured overnight in LB broth supplemented with ampicillin, and then sequenced on both strands using the vector primers M13F-47 and M13R-48 in Invitrogen Inc. . The nucleotide sequences were compared with those in GenBank database using nucleotide BLAST.How to cite this article: Qiao, S. et al. Methanogenesis from wastewater stimulated by addition of elemental manganese. Sci. Rep.5, 12732; doi: 10.1038/srep12732 (2015).


SA) plays an important role in the survival of red blood cells (RBCs), the age‐related reduction in SA content negatively impacts both the structure and function of these cells. We have therefore suggested that remodelling the SA in the membrane of aged cells would help recover cellular functions characteristic of young RBCs. We developed an effective method for the re‐sialylation of aged RBCs by which the cells were incubated with SA in the presence of cytidine triphosphate (CTP) and α‐2,3‐sialytransferase. We found that RBCs could be re‐sialylated if they had available SA‐binding groups and after the re‐sialylation, aged RBCs could restore their membrane SA to the level in young RBCs. Once the membrane SA was restored, the aged RBCs showed recovery of their biophysical and biochemical properties to similar levels as in young RBCs. Their life span in circulation was also extended to twofold. Our findings indicate that remodelling membrane SA not only helps restore the youth of aged RBCs, but also helps recover injured RBCs.Membrane sialic acid ( Circulating RBCs must constantly squeeze through narrow capillaries Regarding RBCs stored in a blood bank, the preservation solution acidifies during storage, and the membrane SA content of RBCs decreases faster than in circulation In addition to the decrease in membrane SA seen in aged cells, when the membrane SA of RBCs is reduced experimentally by enzyme treatment, the biochemical and biophysical properties and survival time of the cells also suffer Sialic acid is an acidic nine‐carbon sugar typically found as the outermost unit of glycan chains on the surface of all vertebrate cells and on secreted glycoproteins i) the old human RBCs aged about 90–120 days in circulation. According to previous researches, they can be separated from freshly drawn venous blood with Percoll density gradient centrifugation as the dense cells in the bottom fraction ii) The RBCs from standard blood‐banked leukoreduced CPDA‐1 human RBC units stored for 21 days (designated B21 cells). (iii) The RBCs from rabbit venous blood stored for 21 days in blood‐bank conditions after drawing from the animal (designated A21 cells).Given the importance of SAs in RBC structure and function and because SA content decreases with cell age, we have suggested that remodelling the SA in the membrane of aged cells would help recover cellular functions characteristic of young RBCs. To test our hypothesis, we incubated aged RBCs with SA in the presence of CTP and α‐2,3‐sialytransferase to induce restoration of membrane SA and then investigated if cell structure, function, and survival improved following re‐sialylation. The aged RBCs defined in here are:  and from 18 male rabbits . Our study protocol was approved by the Ji Nan University Animal Care and Use Committee and conformed to the Chinese Public Health Service Policy on Humane Care and Use of Laboratory Animals.Heparinized venous blood was centrifuged three times at 2000 × g for 10 min. at 4°C with isotonic PBS . Then, the washed RBCs were fractionated with Percoll centrifugation 21 cells.Eight bags of standard blood‐banked leukoreduced CPDA‐1 RBC units were obtained from Guangzhou Blood Services Center, China and stored for 21 days. The RBCs of the units are designated B21 RBCs was incubated with 100 μl NANA , 0.5 ml CTP , and 1.5 ml α‐2,3‐sialytransferase .To incorporate SA, 1.0 ml diluted old RBCs or BMaackia amurensis; Biological, USA) and the surface charge of RBCs was labelled with CdSe/ZnS core‐shell quantum dots  as described previously The zeta potential of the cells was measured with a Zeta PALS potential analyser  at 37°C. Sialic acids on the RBC membrane were labelled with fluorescein isothiocyanate (FITC)‐MAA , as was the level of 2,3‐DPG in RBCs .Adenosine triphosphate (ATP) enzymatic activity of blood cytolysates including that of both NaOryctolagus cuniculus, provided by the Institute of Laboratory Animals, Jinan University) were randomly divided into three groups (six rabbits/group). Five millilitres of blood was taken from the ear artery of each rabbit and collected into a CPDA‐1–containing blood bag. Cells were washed  two times with normal saline (0.9% NaCl), producing leukoreduced RBC samples.To determine RBC survival, 18 New Zealand White rabbits (1), RBCs were incubated with 350 μg FITC  for 30 min., and then washed  three times with normal saline to eliminate residual FITC. The labelled RBCs were then re‐suspended to their original volume in normal saline and autologously transfused into the rabbits through the marginal ear veins. After re‐infusion of the labelled RBCs, blood samples (5 μl per rabbit) from the marginal ear vein were collected into heparin‐containing tubes after 1 and 24 hrs and then every other day. The samples were washed with normal saline , suspended in 2 ml normal saline, and assayed with a flow cytometer  to determine the percentage of FITC‐labelled RBCs. The half‐life of the labelled RBCs was the number of days at which the survival rate was 50%.For the first group (G2) and group 3 (G3), the blood samples were stored for 21 days and then prepared as leukoreduced RBCs. The G2 RBCs were incubated with 2.5 ml SA , 1.5 ml α‐2,3‐sialytransferase , and 0.5 ml CTP (7.4 mM) for 4 hrs. Then the cells were washed  two times with normal saline to eliminate residual SA. Similarly, after labelling with FITC, the G2 and G3 RBCs, the latter of which were not incubated with SA, were re‐infused into the animals. The sampling and assay procedure were the same as that for G1 RBCs.For group 2 . P‐values were obtained by comparison of cells without or with incorporation of SA.Statistical analyses were performed with 21 cells  and major axis (7.43 ± 0.22 μm) of O cells were significantly smaller (P < 0.05) than the values for young RBCs (46.42 ± 4.61 μm2 and 7.85 ± 0.55 μm respectively). Aggregation of O cells was also observed. After incubation with SA, however, the O cells assumed a discocyte shape similar to young cells and aggregation was no longer observed. Similarly, after incubation with SA (80 μg/ml), B21 cells recovered their morphology, i.e. changed from 40.07 ± 4.16 μm2 for contact area and 7.31 ± 0.65 μm for major axis to 44.09 ± 5.68 μm2 and 7.88 ± 0.54 μm, respectively.As seen in Figure Kc, of the three types of cells  as a function of SA concentration. As described previously Kc is inversely proportional to membrane deformability. The Kc of both O and B21 cells decreased with SA concentration and at 80 μg/ml the Kc was the same as that of young cells. To determine if the cells had recovered sufficient deformability to travel through capillaries in circulation, we aspirated the cells into a capillary with an inner diameter of 1.8 μm. The time needed to aspirate the SA‐restored cells into the capillary under a negative pressure of 1500 Pa was 0.66 sec., which was almost the same as that of young cells (0.67 sec.) but significantly shorter than the time for O cells . Similar results were obtained for re‐sialylated A cells.Figure 2‐binding capacity of Hb in RBCs, we performed confocal Raman micro‐spectroscopy on the cells. Figure 830/I850 in the Raman signal of old RBCs was less than 1, indicating that Tyr was exposed. The bands at 1266 and 1280 per cm represent the unordered coil and the α‐helix protein conformations of amide III respectively 2.To determine if restoration of SA affected the OThe distribution of intracellular Hb was also restored after incorporation of SA Fig. C. In con2‐carrying capacity of aged cells, we used Raman microscopy to measure the speed at which cells transitioned from a deoxygenated (T) state to an oxygenated (R) state via interrelated states that are named M1, M2, M3 and M4 2 had recovered.To determine whether SA remodelling improved the O+, K+‐ATPase activity, Ca++Mg++‐ATPase activity and the 2,3‐DPG level of SA‐restored B21 cells. The activity of Na+, K+‐ATPase and Ca++Mg++‐ATPase and the 2,3‐DPG level in the aged cells increased with SA incorporation  again by Percoll centrifugation, and the total number of cells was the same as in the freshly drawn blood sample unit 21 cells can survive for longer time in blood‐bank condition.According to our previous study 1) of animal experiments for RBC survival, RBCs from freshly drawn blood were labelled with FITC and immediately injected back into the animals. In the other two groups, RBCs were separated from animal blood that had been stored for 21 days; one group (G2) received cells that were incubated with SA (40 μg/ml), and the other group (G3) were not treated with SA. Approximately 94% of G2 cells survived after 24 hrs, which was similar to RBCs of G1 but significantly higher than cells that were stored for 21 days without SA incubation , the half‐life was significantly longer than G3 cells , indicating that, after SA remodelling, the SA‐restored senescent cells showed improved viability and an extended lifespan.In the first group . The animals in group GKc decreased from 1.85 ± 0.06 × 10−19 J to 1.68 ± 0.04 × 10−19 J , and about 32% of abnormally shaped cells returned to a normal discocyte shape. Thus, remodelling membrane SA also helped restore the structure and function of some unhealthy RBCs.To determine whether viability could be restored to unhealthy cells by treating them with SA, we performed an experiment with RBCs from patients with anaemia. Upon remodelling of SA (30 μg/ml), the zeta potential of the unhealthy cells increased from ¦−20.8¦ ± 0.8 mV to ¦−26.3¦ ± 0.6 mV, which was very close to the value of a control with normal RBCs (−27.1 ± 0.8 mV). Correspondingly, the According to the results presented in Figures 21 cells and A21 cells. This suggests that the membrane SA only can be restored to a certain value and re‐sialylation requires unoccupied SA‐binding groups on the cell surface. To prove it, we incubated young RBCs from freshly drawn human blood with NANA. Although a very high concentration (≥80 μg/ml) of SA was used, the zeta potential of the cells increased just slightly from ¦−30.2¦ ± 1.2 mV to ¦−31.7¦ ± 0.8 mV and then plateaued as the behaviour of re‐sialylated‐aged cells shown in Figure From Figure As demonstrated previously that SAs govern the morphology, membrane deformability, oxygenation capacity and even the structure and distribution of the intracellular Hb molecules in human RBC +, K+‐ATPase and Ca++Mg++‐ATPase activities and the 2,3‐DPG level because that recovery of band 3 conformation improves membrane permeability. Since membrane permeability correlates positively with ATPase activity/ATP concentration in RBCs, activation of the ion pump can significantly increase intracellular ATP The recovery of the band 3 conformation after restoration of membrane SA in SA‐restored aged RBCs may be also a factor that helped restore the NaOur results show that remodelling membrane SA has a remarkable effect on aged RBCs, restoring their youth, extending their lifespan, and promoting recovery of cells that had been stored for a long time or that were dysfunctional. These findings have broad biological and medical implications for the recovery of aged and diseased cells and may be applicable to blood‐banked RBCs to prevent ‘storage lesion’, which was believed as one of the most critical issue facing transfusion medicine The authors have declared that no competing interests exist.YXH conceived and designed the experiments. WWT, DW, LLK, XYC performed the experiments. YXH and ML contributed analysing the data. YXH wrote the paper. YXH would like to thank Dr. J Mehrishi for the useful discussion on the effect of SA.


The number of quintuple Sb2Te3 2D crystal layers dictates the strain in the GeTe layers and consequently its diffusive atomic disordering. By identifying four critical rules for the superlattice configuration we lay the foundation for a generalizable approach to the design of switchable van der Waals heterostructures. As Sb2Te3–GeTe is a topological insulator, we envision these rules enabling methods to control spin and topological properties of materials in reversible and energy efficient ways.Strain engineering is an emerging route for tuning the bandgap, carrier mobility, chemical reactivity and diffusivity of materials. Here we show how strain can be used to control atomic diffusion in van der Waals heterostructures of two-dimensional (2D) crystals. We use strain to increase the diffusivity of Ge and Te atoms that are confined to 5 Å thick 2D planes within an Sb Strain engineering allows design of materials with tailored properties. Here, the authors show that strain can be used to control atomic diffusion in Sb2Te3-GeTe superlattices, and they propose general design rules to enable atomic switching functionalities in van der Waals heterostructures. A systematic design methodology would accelerate the optimization of Sb2Te3–GeTe heterostructures, phase change materials and switchable topological insulators2Te3–GeTe heterostructure and allows diffusive atomic disordering within the GeTe layers. In practice we demonstrate that this Ge–Te diffusive switching is controlled by the thickness of the Sb2Te3 layers and confined to an interface just 5 Å thick.van der Waals (vdW) heterostructures composed from layers of two-dimensional 2D) atomic crystalsD atomic 2 2 ref. , respect2Te3–GeTe phase change materials. This may be due to the misbelief that reversible switching in these materials is only possible by a melt–quench process, which would cause strain relaxation. Recently, a number of demonstrations have shown that switching in Sb2Te3–GeTe materials is possible without melting the crystal structure. For instance, the optical properties of the Ge2Sb2Te5 alloy can be switched without transiting through the molten phase2Te3–GeTe vdW heterostructures2Te3–GeTe vdW heterostructures.Strain engineering provides a conceptually simple means to control the properties of materials at an atomic level. Furthermore, it is well suited to computer-aided atomistic design of materials using tools such as density functional molecular dynamics (DF/MD). In contrast, modelling doped materials require a large simulation cell and hundreds of atoms to reach the low dopant concentrations that are typically used in experiments. Consequently, simulating doped materials is extremely computationally expensive. Models of strained crystals do not suffer from this problem. It is, therefore, surprising that strain engineering has not been applied to the development of Sb2Te3–GeTe superlattice structures have been designed to promote the movement of interfacial Ge atoms through a plane of Te atoms2Te3–GeTe superlattices, the electrical resistivity increases by several orders of magnitude yet the superlattice remains crystalline. This led to the suggestion that the material undergoes a crystal–crystal transition2Te3 superlattice-like structures (SLL)2Te3 layers within the as-deposited SLL are relatively thick amorphous films that crystallize at different temperaturesCrystalline Sb2Te3-based superlattices by changing the composition of the other superlattice layers18192Te3–GeTe superlattice such that the GeTe layers disorder at a temperature substantially lower than the melting temperature of the whole superlattice. Strain engineering is a more general approach to materials design than compositional tuning. Moreover, the approach can in principle be used to add switching function to vdW superlattices composed of other 2D crystals.The aim of this work is to establish a set of general rules that can be used to design and optimize phase transitions in vdW superlattices. Others have tried to further optimize switching in Sb2Te3–GeTe superlattice is dominated by the vertical movement of Ge atoms through a plane of Te atoms. By calculating the activation energy for a single Ge atom to switch through the planar Te layer using the nudged elastic band transition state search method, which is illustrated in 2Te3 and GeTe layers are stable but in reality a high temperature, close to the structure's melting point, is required for a significant proportion of the interfacial atoms to undergo atomic switching.Recently Yu and Robertson2Te3 has a larger in-plane lattice constant than GeTe. To assess the tensile strain applied to the GeTe layers of Sb2Te3–GeTe superlattices, we grew different superlattice structures with the Sb2Te3 layer thickness varying from 1 to 4 nm, whilst the GeTe layers' thickness was fixed at 1 nm. The superlattice lattice parameters were measured by X-ray diffraction in a symmetric geometry. Only the (00L) peaks were present in the diffraction patterns thus indicating highly textured, layered superlattice films, see 132Te3 and GeTe layers relative to their bulk values, see 2Te3 layer thickness is increased, the biaxial strain in the GeTe layer increases from 0.72 to 2.16%. By comparing these measured strain values with the activation energy, which is shown in 2Te3 layers. Therefore a lower switching energy is to be expected for Sb2Te3–GeTe structures with thicker Sb2Te3 layers.Sb2Te3–GeTe superlattice heterostructure, the GeTe crystal is strained and therefore distorted with respect to the bulk GeTe crystal. We therefore investigated the effect of biaxial strain on the stability of the GeTe layers within the Sb2Te3–GeTe heterostructure by DF/MD simulations. The crystallinity of the individual GeTe and Sb2Te3 layers was calculated as a function of time. The crystallinity evolution of the individual Sb2Te3 and GeTe layers for the 2% biaxially strained superlattice is shown in 2Te3 and the GeTe layers show an abrupt decrease in crystallinity at a similar melting temperature. However, the GeTe crystallinity gradually decreases as the temperature increases above 600 K. In effect, premelt disordering starts to occur in a 5 Å thick GeTe 2D plane at a temperature 300 K below the melting temperature of the whole structure. Although melting is stochastic by nature, there is a clear trend that increasing biaxial strain decreases this GeTe premelt disordering temperature, as shown in 2Te3 layers' thickness.It is clear from 2Te3–GeTe crystalline superlattice samples into a low reflectivity state. The minimum laser fluence required to write a low reflectivity spot into the film is shown as a function of strain in 2Te3 layer thickness reduces the fluence necessary for switching the optical properties of the structure. For reference, the measured switching energy for cubic Ge2Sb2Te5 is also shown. It is worth noting that the superlattice composed of 1 nm thick Sb2Te3 layers and 1 nm thick GeTe layers has the average composition Ge2Sb2Te5. However, its threshold switching energy is lower than the Ge2Sb2Te5 alloy, which shows that the superlattice structure can be used to design and optimize switching in Sb2Te3–GeTe-based materials. Moreover, the GeTe layers in superlattices composed of 1 nm thick Sb2Te3 layers and 1 nm thick GeTe layers are subjected to almost 1% biaxial strain relative to the unstrained 40 nm thick GeTe film, see We used femtosecond laser pulses to measure the energy required to switch the superlattices into a low reflectivity state. Single 30 fs laser pulses at a wavelength of 800 nm were used to switch 40 nm thick SbTo confirm the reversibility of the switching process the optical reflectivity of the laser switched marks was measured before and after annealing the samples at 220 °C. The optical reflectivity for all the samples typically increased by 15% after annealing such that the switched areas were indistinguishable from the background crystalline film. This indicates the reversibility of the GeTe disordering process .2Sb2Te5 showed that the melting process is typically complete in 5 ps 2Te3 (891 K)2Te3 layer is increased, the GeTe lattice is put under biaxial tensile strain and consequently the Ge–Te resonant bonds are weakened and the disordering temperature of the GeTe layers is lowered. In contrast, the lattice parameter of the Sb2Te3 layers tend to the bulk value and therefore the layers remain crystalline, see 2Te3 and biaxially tensile strained GeTe, which effectively increases the stability of the Sb2Te3 layer whilst decreasing the stability of the GeTe layer. This situation is illustrated in 2Te3 thickness increases the strain in the GeTe layer, destabilizing it and promoting premelt disordering. The reduction in the laser energy used to disorder the GeTe layers, which is shown in The GeTe premelt disordering, which is shown in 2Te3 layers can remain crystalline during the GeTe disordering. During the 43 ps simulation Sb2Te3 atoms retained their crystallographic positions. Disordering is confined to the GeTe layers that are less than 5 Å thick—a 2D phase transition occurs. The final ‘switched' structure is shown in 2Te3 layers in the unstrained superlattice are unstable and both the GeTe and Sb2Te3 layers inter-diffuse, see 2Te3–GeTe vdW heterostructures can be thermally stimulated by premelting the GeTe layer at temperatures lower than the melting point of Sb2Te3. Increasing the thickness of the Sb2Te3 layer increases the biaxial strain in the GeTe layer and causes a related increase in the atomic switching probability and a decrease in the switching energy. The Sb2Te3 layers experience little distortion during the switch and their rigidity is crucial for the stability of the whole superlattice structure. As such, Sb2Te3 presents a versatile scaffold to strain engineer the properties of GeTe. We conclude that in-plane biaxial strain significantly enhances diffusive atomic disordering within the GeTe layers of Sb2Te3–GeTe superlattices.From these results we conclude that diffusive Ge–Te atomic switching in Sb2Te3–GeTe heterostructures have been developed. However, we believe that the applicability of strain engineering can be generalized and extended to other vdW bonded heterostructures of 2D chalcogenide crystals and therefore we highlight the following rules that can be used to design similar diffusive atomic switching heterostructures: First, the switching material (GeTe) in the heterostructure must exhibit resonant bonding such that its structure is sensitive to distortions and strain can be used to modify the disordering energy. Second, the scaffold material (Sb2Te3) in the heterostructure must be stable over the temperature range that the switching material exhibits premelting. Third, the scaffold material must have a larger bulk lattice parameter, a, than the switching material. Fourth, it must be possible to grow the vdW heterostructure superlattices from 2D crystals of the switching and scaffold materials by vdW heteroepitaxy such that the layers strain one another.Herein, diffusive atomic switches based on strain-engineered Sb2GeTe3 2Te3–GeTe model was built. For the DF/MD runs energies were calculated at the Γ point of the Brillouin zone (k=0), which led to an accuracy of 50 meV per atom relative to a 5 × 5 × 5 k-point mesh. We used a plane-wave basis with 220 eV cutoff. The energy precision for the self consistent field calculation was set to 10−5 eV. The temperature of the model was controlled by velocity rescaling. The superlattice unit cell dimensions were estimated by running 10 ps of MD at 300 K with different cell dimensions. The 300 K relaxed cell dimensions were then found by selecting the structure with the lowest square-average stress tensor diagonal elements over the last 5 ps of the trajectory. This cell was then elongated by the desired strain amount in the xy-plane, and similar 10 ps simulations were performed to find the relaxed z-length while keeping the lateral strain.To model the effect of biaxial strain on diffusive atomic transitions, an 81 atom Sbet al.2Sb2Te5  order parameter of Steinhardt Te5 ref. . The BO rij is the vector between atoms i and j, N(i) is the number of neighbours for atom i, and Nb includes the atom i and its neighbours. The Q4(i) value for ideal, vacancy-free rock-salt lattice is 0.764. To compensate for thermal fluctuations we defined a crystalline atom as having a Q4(i) magnitude greater than 0.6. This method results in a good agreement with the atoms marked as crystalline by the order parameter and the atoms that look crystalline in visualizations.where 2Te3–GeTe superlattice was studied with a temperature ramp for each strain. The initial temperature was 450 K, and it was increased at 1.97 K ps−1 until the structure melted. The initial velocities were random, and scaled according to the temperature. The layer crystallinities were calculated as an average over all the atoms occupying the space initially occupied by each layer, and over 1 ps (1.97 K) windows.The premelting in the Sb2Te3 model cell with ultra-soft pseudo-potentials. The plane-wave basis cutoff energy was set to 230 eV. The atomic positions and the cell shape was relaxed to the ground state using the BFGS−6 eV per atom. The maximum force on the atoms was less than 0.01 eV Å−1. The energy was calculated at the gamma point. The atomic positions of the switched structure were also relaxed using the same criteria as listed above. The simulation cell volume was fixed with the non-switched volume of 2,501.63 Å3. The nudged elastic band method was then used to calculate the activation energy for a single Ge atom to switch into the vdW gap. A linear synchronous transit (LST) search was used to initially approximate the barrier height. The activation energy was then refined by cyclically repeating a quadratic synchronous transit (QST) maximization with a conjugate gradient minimization until the stationary point of the barrier was foundThe activation energy for a single Ge atom to switch into the vdW gap was calculated with the DFT code CASTEP2Te3, 2 nm—GeTe, 1 nm) and  superlattice structures to find their in-plane lattice parameter, rather than modelling the larger lattice parameter by straining the  structure, which we did here.Note that the DFT energy of our models was not corrected for the inter-block vdW interaction. Our strain-engineered switching model is based on the GeTe layer disordering process being dominated by the in-plane separation of Te–Te bonds rather than the out-of-plane vdW interactions. 2Te3, x nm—GeTe, 1 nm), where x=1, 2 and 4, superlattice films for laser switching measurements were grown on Si (100) substrates using layer-by-layer sputter deposition. Before the superlattice deposition the native oxide was removed from the Si substrate using a 30 W RF plasma for 60 min at a substrate holder temperature of 300 °C. A 10-nm thick Sb2Te3 buffer layer was then grown on the Si surface before the deposition of the superlattice film. The samples were grown in an Ar atmosphere of 0.5 Pa, whilst the base pressure of the vacuum chamber was better than 2.5 × 10−5 Pa. The substrate holder temperature was held at 300 °C during the superlattice growth. For all samples the total film thickness was kept constant at 40 nm and the composition was confirmed by energy dispersive X-ray (EDX) spectroscopy. The lattice parameters  of the superlattices were determined by Rietveld refinement. The in-plane biaxial strain applied to the GeTe and Sb2Te3 layers is dictated by the superlattice in-plane lattice parameters, and this was estimated by aGT and aST are the in-plane lattice constants for bulk GeTe and bulk Sb2Te3 in a hexagonal setting. To avoid encapsulation induced stressesThe .Supplementary figures 1-9, Supplementary table 1, Supplementary notes 1-6 and Supplementary references.


N). A two-color resonant two-photon ionization scheme is applied to study the Cu 2P1/2 ground state transition. The absorption is strongly broadened for Cu atoms submerged inside helium nanodroplets and a comparison with computed literature values is provided. An observed ejection of the dopant from the droplet is triggered upon excitation, populating energetically lower states. The formation of Cun clusters up to Cu7 inside helium nanodroplets was observed by means of electron impact ionization mass spectroscopy.Mass and optical spectroscopic methods are used for the analysis of copper (Cu) atoms and clusters doped to helium nanodroplets (He It is well established that alkali atoms reside on the droplet surface N can be extracted from absorption broadenings and line shifts n − 1)d10ns electron configuration of coinage metal atoms can be considered alkali like, so these species are well suited for spectroscopic analysis and were already doped to various matrices. In He droplet experiments, preferably silver was used as a single atom dopant n clusters and organic molecules n clusters Most experimental and theoretical studies of metal doped HeN system to provide comparison to theoretical studies ω1 + ω2 resonance ionization spectroscopy (RIS) scheme is used to study the excitation with one photon utilizing a resonant transition from the ground state and a second photon providing the energy to ionize, a scheme that was also used in free Cu atom spectroscopy N and mass spectroscopic observation of small Cun clusters formed inside the droplets.We were able to measure the absorption of the Cu–He2N are formed by a supersonic expansion of He gas (purity 99.9999%) through a cooled nozzle  into vacuum. The He condenses to form clusters following a log-normal size distribution with, under these conditions, typical maxima in the distribution of r = 4.3 − 2.5 nm (mean droplet size d = 300 μm) to shape a HeN beam traveling toward a pickup oven located in a separately pumped vacuum chamber where the HeN statistically pick up one or more Cu atoms. The resistively heated Cu evaporation source consists of a tungsten wire heated alumina (Al2O3) coated crucible covered with a slitted molybdenum lid with a slit length of 25 mm, arranged parallel below the droplet beam. Gas phase Cu atoms are emitted from the slit and intersect the HeN beam at right angles, hence the crossed beam geometric layout – including 5 small apertures to collimate the droplet beam – ensures that no free atoms reach the detection region 1.4 m downstream. For the doping with single Cu atoms, oven temperatures of typically 1000–1100 °C are necessary in this setup. For the formation of Cun clusters inside HeN, the experimental conditions are chosen to favor the pickup of, on average, more than one Cu atom per droplet, meaning higher crucible temperatures (1100–1300 °C) and large droplets (N beam with a laser beam either at right angles for photoionization (PI) or antiparallel for beam depletion (BD) measurements. For analysis of the embedded Cun clusters by means of electron impact ionization mass spectroscopy, the QMS is equipped with a crossed beam electron bombardment ion source. Here, the HeN beam is chopped for differential counting with a home-built two channel counter.The experimental setup follows the design of a HElium NanoDroplet Isolation (HENDI) spectroscopy apparatus described in detail in previous publications λ = 308 nm) is coupled out of the dye laser pump beam and guided toward the HENDI apparatus simultaneously with the tunable dye laser beam. Both are focused and overlapped inside the ionization region of the QMS . The R2PI and BD spectra presented are exclusively recorded at 63 u, the mass of the most abundant Cu isotope. For the observation of photoionized fragments, the mass filter is scanned while ionization is accomplished with fixed laser wavelengths. Overall, this setup allows us to perform resonance ionization spectroscopy of Cu–HeN complexes with mass selective ion detection and electron impact ionization mass spectroscopy.For the Cu excitation in the UV spectral region, the radiation from an excimer  pumped dye laser  with ∼25 ns pulse duration and 100 Hz repetition rate is frequency doubled with a KDP crystal . Excitation wavelengths are determined from the fundamental wavelength measured with a wavemeter (Coherent Wavemaster). For Resonant two Photon Ionization (R2PI), part of the excimer laser beam (2S1/2) electron configuration is [Ar]3d104s. In principle, the ground state absorption measurement follows the simple R2PI excitation scheme  and D2 (−1) 2P2P104p) states, respectively. The Cu II limit lies at −1 which cannot be reached from the Cu★ intermediate 2P−1 and is thus accomplished by the absorption of a XeCl laser photon (−1).The Cu ground state (heme see  of Ref. 33.1−1 to 32 120 cm−1 while the second laser is kept constant at 32 468 cm−1. In the upper panel of 12 (dotted blue curve) and CuHe100 (dashed red curve) 2P1/2 transition energy region. The ions are detected exclusively at the mass of the most abundant 63Cu isotope. Most obviously, it consists of (1) sharp peaks at the catalogued Cu gas phase transition energies which are (2) broadened on their high energy side by up to ∼200 cm−1 wide shoulders. These features are discussed below, while first the broad structure (3) stretching over ∼900 cm−1 between −1 and −1 is treated.A number of characteristic absorption features is observed with R2PI, as one laser is scanned from 30 470 cm−1 is assigned as 2P1/2 excitation of Cu inside the droplet, in reasonable agreement with the calculation of Cargnoni and Mella 100. This is characteristic for outer shell transitions of atoms surrounded by the droplet. Cavities are formed around the dopant and a qualitative understanding of spectral line broadening and shift is provided by the size-dependent energy of the atomic bubble state. During the orbital expansion of a vertical electronic excitation the He atoms inside the cluster cannot readjust their positions In N is often described using the model by Ancilotto et al. λ≥ 1.9. The calculated value of λ = 2.9 N and the distinct Cu–He well depth of −28.4 μhartree (−0.77 meV) estimated with ab initio ground state pair potentials The solvation of metal atoms inside Her = 3.9 nm). The observed broad and blueshifted absorption (3) agrees well with the simulated absorption for a cluster size of n = 100 He atoms The first laser excitation is accomplished while the Cu atom resides inside a droplet with N neglect any photon-induced secondary effects described below. The BD spectrum shall be compared to the computed CuHe100 (dashed red curve) vertical excitation spectrum representing the in-droplet excitation best. The onset of the droplet broadened structure lies approximately 100 cm−1 to the red of the computed absorption, reaching the maximum absorption level already at 30 800 cm−1. So we attribute the deviation from computational results Beam depletion measurements  at and (2) in the vicinity of the free atom ground and excited state transitions. As described above, the experimental setup prevents free atoms from reaching the PI-region from the evaporation source. We hold a photo-induced ejection mechanism responsible for the production of un- and weakly perturbed Cu atoms, like it was predicted by F. Cargnoni and M. Mella in the form of a “spit out” from the droplet following the 2P1/2 excitation L = 0, ΔS = 1) should be dipole forbidden but has a low free atom transition strength 4D3/2 transitions points toward a strong population of the metastable 2D3/2 state. Exciplexes which form when one or several host atoms (helium) penetrate into the nodal region of the excited valence electron's density distribution 2Σ+, 2Π1/2, 2Π3/2 and the 2Σ+, 2Π1/2, 2Π3/2, 2Δ3/2, 2Δ5/2 potential energy curves of the Cu–He diatomic which converge asymptotically to the atomic 2P and 2D doublets nP orbitals. The elucidated mechanism might also be extended to nonresonant excitation in the vicinity of the resonant state. One excimer laser photon may provide excitation near the resonant transition (see N beam were never observed by us and are not expected due to our “crossed beam” pickup-geometry. The sharp lines (1) at the wavenumbers of the free Cu 2P1/2 and 2P1/2 transitions indicate that within the pulse length of our laser (∼25 ns) a Cu atom can be excited inside the droplet, be ejected from the droplet, relax to the ground state, and be resonantly ionized through the two 2P states. Furthermore, the spectrum in 12, which are nevertheless shifted by about 130 cm−1 to the blue, about the same amount as the shift for simulated Cu–He100 against our depletion band. We conclude that our measured bands (2) derive from small Cu–Hen  complexes ejected from the droplet along with the free atoms mentioned above. While our ion mass scans only show (CuHe)+ and (CuHe2)+, recent calculations of Cargnoni et al. ★(2P) state is capable of binding up to 5 He atoms. Simulations of excitation spectra of the complexes CuHe to CuHe5 would shine more light on our observations. The overall agreement between the calculations −1 is similar as in the case of Ag atoms solvated in HeNWhile the broad absorption band originating from Cu inside Hetion see  and lead3.2N from Cu2+ to Cu7+. The formation of Cun clusters is confirmed by the distinct patterns according to the isotopic composition of 63Cu and 65Cu. A comparison with the calculated binomial distribution for the Cu7+ cluster is shown in the lower panel of 7 cluster 7 cluster formation. This is realistic since, according to the log-normal distribution m+ compounds and their fragments. An investigation of the electronic properties of such metal–water clusters in the gas phase was recently reported N fragment ions.N. CrCu, CrCu2, Cu2Cr, and CrCu3 were successfully detected by means of electron impact ionization mass spectroscopy. These species are promising candidates for spectroscopic investigation on HeN, as only little data is available Successive doping of the droplet beam with Cr 4N by means of laser ionization spectroscopy with mass selective detection and compare them to computational results from literature −1 blueshifted absorption band is observed as depletion spectrum and is assigned to Cu atoms solvated inside the droplets. Predicted photoinduced ejection was observed including the relaxation to the ground and intermediate 2D states together with the formation of small Cu–Hen clusters. For a better comparison with calculations, the computation of absorption spectra for Cu–Hen clusters with less than 12 He atoms or for Cu atoms residing on the droplets surface would be helpful.In this work, we examine the absorption and relaxation mechanisms of Cu atoms embedded inside Hen clusters and Cun(H2O)m+ compounds inside HeN was shown for up to seven Cu atoms and evidence is provided that a significant fraction of the droplets survive this cluster formation. This will allow systematic spectroscopic studies of electronic spectra of these systems.The successful formation of small Cu


Monolayers of transition metal dichalcogenides can exist in several structural polymorphs, including 2H, 1T and 1T′. The low-symmetry 1T′ phase has three orientation variants, resulting from the three equivalent directions of Peierls distortion in the parental 1T phase. Using first-principles calculations, we predict that mechanical strain can switch the relative thermodynamic stability between the orientation variants of the 1T′ phase. We find that such strain-induced variant switching only requires a few percent elastic strain, which is eminently achievable experimentally with transition metal dichalcogenide monolayers. Calculations indicate that the transformation barrier associated with such variant switching is small , suggesting that strain-induced variant switching can happen under laboratory conditions. Monolayers of transition metal dichalcogenides with 1T′ structure therefore have the potential to be ferroelastic and shape memory materials with interesting domain physics. The atoms in two-dimensional transition-metal dichalcogenides can arrange into a number of different structures, or polymorphs. Here, the authors use first-principles calculations to show that one such polymorph, 1T', can exhibit a large mechanical response to external applied strain. In the 1T phase, the W atoms arrange in 2D triangular lattice, which is sandwiched between two Te atomic layers. The 2D primitive cell of the 1T phase is a 120° rhombus with side length t0. Due to Fermi surface nesting induced Peierls distortiona × b, which corresponds to the We use WTeh1 and h2 of the 1T supercell can be written as x and y directions labelled on H0={h1,h2} can be constructed, where h1 and h2 are treated as column vectors, that is,The spontaneous transformation strains associated with the 1T to 1T′ transformation can be compared between the three variants based on the H1, H2 and H3, respectively. These new supercell matrices can be related to the original supercell matrix by transformation matrices Ji, which map the undistorted supercell to the distorted supercells. Namely, Hi=JiH0, where the subscript i stands for the i-th orientation variant. The transformation strain matrices ηi associated with different variants can then be calculated from Ji based on the definition of Green-Lagrange strain tensor:After transforming to the 1T′ phase, the distorted supercell matrix corresponding to the O1, O2 and O3 variants will be denoted by I is a 2 × 2 identity matrix. The 2D transformation strain tensor ηi has the following symmetric form:Here, the superscripts −1 and T denote matrix inversion and transposition, respectively. ɛxx and ɛyy are the tensile/compressive strain along x or y direction, and ɛxy is the shear strain component.where Hi for different MX2 monolayers are tabulated in ηi can be evaluated, which are listed in 2 monolayers, the transformation strain matrices form 1T to 1T′ areWe have employed DFT calculations to obtain the equilibrium supercell vectors and the relaxed atomic coordinates of the O1, O2 and O3 variants, resulting from the distortion of 2 is 1T′, it is informative to directly compare the distorted supercell of the three variants by computing the relative supercell strain associated with the transformation from one variant to another. This can be carried out again using the supercells of the three variants derived from the common i to j. Calculations based on the same definition of strain tensor as in The difference in transformation strain between the three variants of 1T′ suggests that one may switch the relative thermodynamic stability between different variants by applying suitable external mechanical stress. Since the equilibrium structure of WTeIt then follows that, starting with the O1 variant of 1T′ in a strain-free state, after imposing an external strain of magnitude 2 monolayer, we have used DFT to calculate the potential energy surfaces of the three variants of 1T′ as a function of 2D supercell dimensions. We first investigate the possibility of mechanically switching the O1 variant to O2 or O3 variant by applying biaxial strain to the system, again using WTe2 monolayer as an example. The strain-free 2 × 2 supercell of the O1 variant, derived from the distortion of the aforementioned a × 2b within the x–y plane of 2D monolayer. We adjust the dimensions of the supercell along x and y directions independently, with the values of a and b range from −10 to 10% of engineering strain at an equal step of 2%. At each pair of , the atomic coordinates within the supercell are relaxed. We also compute the energies of O2 and O3 variants when their supercell dimensions are fixed to be the same as O1. The energies U of all three variants are computed on a 11 × 11 grid in the  space, giving a total number of 121 data points distributed evenly around the equilibrium lattice constants of the O1 variant. Smooth potential energy surfaces are then constructed by approximating the intermediate values of U using 2D spline interpolation, which allows us to directly compare the relative energetic stability of the O1, O2 and O3 variants in the full  space. In addition, the U for the 2H phase is computed for comparison, as a previous study indicates that strain-induced phase transformation between the 2H and 1T′ phases can happen in MX2 monolayersTo study in detail the relative thermodynamic stability of different variants when external mechanical deformation is imposed on a 1T′–MXa,b) space are determined. The result is shown in 2 monolayers10a,b) space are essentially the same. This can be rationalized by the fact that the supercells of both variants can be derived from the distortion of the y direction in the 1T phase, as can be seen from y axis, the O2 and O3 variants are still mirror images of each other and have the same energy. We however expect that shear strain, which breaks the mirror symmetry, can distinguish the energies of all the three variants of 1T′. Indeed, ɛxy of magnitude >3.5% is imposed on the O1 variant, O3 becomes the lowest-energy variant within the strained supercell. If the sign of ɛxy is reversed, then the O2 variant has lower energy than both O1 and O3.After obtaining the potential energy surfaces for all the three variants of 1T′ as well as the 2H phase, the lowest-energy variants/phases in the  space as the lowest-energy phase. This result is different from the study by Duerloo et al.2 monolayers, as the authors did not take into account the existence of orientation variant degrees of freedom in the 1T′ phase.a,b) space is by applying tensile strain along the a axis of the O1 variant, which is the direction of dimerized metal-atom chains, while simultaneously applying compressive strain along the b axis. It is however known that 2D MX2 monolayers usually cannot sustain large compressive strain due to compression-induced buckling response and formation of ripplocations2 monolayers can withstand tensile elastic strain as large as 10% before mechanical failure102 by uniaxially stretching it along the a axis, which is the direction of dimerized tungsten atoms. This axis can be identified by mechanical cleavage or by the anisotropic response to external fields that is expected for the low-symmetry 1T′ structure2 monolayers, including MoS2, MoSe2, MoTe2, WS2 and WSe2. The results are very similar to WTe2, indicating that strain-induced switching of thermodynamic stability between different orientation variants is generic to MX2 monolayers with 1T′ structure.In F=E—TS, where E is internal energy that includes both potential energy U and kinetic energy, T is temperature and S is entropy. Because all the three variants O1, O2 and O3 belong to the same 1T′ structure, and because the entropy of solids  is relatively insensitive to small deformation, we can use the potential energy U of different variants, computed by DFT at zero temperature, to compare the free energies of different variants at ambient conditions. In 2 when it is uniaxially stretched along the a axis. Consistent with typical experimental set-ups for uniaxial deformation, the stress of the supercell along the b axis is relaxed to zero. This corresponds to free boundary, or zero stress (σy=0) condition along the b axis. In a axis and the same boundary condition along the b axis. A common tangent can be constructed between the energy curves of O1 and O2/O3, which intersects the two curves at uniaxial strains equal to 1% and 4%, respectively. Between these two values, the system can lower its energy by existing in a state where both O1 and O2/O3 variants coexist. This indicates that the formation of O2/O3 variants becomes thermodynamically favourable when the uniaxial strain along the a axis of O1 is as low as 1%.We emphasize that the strain at which the potential energy surfaces of different variants intersects is not the same as the strain at which variant switching becomes thermodynamically favourable. The system can minimize its free energy by choosing a state where different variants (or phases) coexist, akin to the two-phase region in chemical-composition phase diagrams. Under constant temperature and fixed external strain (supercell dimensions), the thermodynamic potential that determines the relative variant/phase stability is the Helmholtz free energy 2 monolayers. Our results suggest that it becomes thermodynamically favourable for the O1 variant of 1T′–WTe2 monolayers to switch to the other two variants when applying uniaxial strain around 1% along the direction of dimerized tungsten atom chains. However, if the kinetic barrier associated with variant switching is too high, such variant switching may not occur under normal experimental conditions and timescale, and the materials would still not be ferroelastic. We have therefore computed the transition barrier associated with the switching between the O1, O2 and O3 variants using climbing image nudged elastic band (NEB) method2 monolayer is shown in Up to now, we have only considered the thermodynamic aspects of variant switching in the 1T′–MX2 monolayers, and the barriers obtained are even lower than 1T′–WTe2 monolayers. The transition barriers of variant switching are significantly lower than the barriers of phase transition between the 2H and 1T′ phase2 monolayers at the equilibrium lattice constants of the 2H phase. The much smaller transition barriers associated with the variant switching within the 1T′ phase as compared with the 1T′ to 2H phase transition has an intuitive geometric explanation. Variant switching between the orientation variants of 1T′ phase only involves the distortion of M–X octahedral coordination, while the 1T' to 2H phase transition requires the complete change of M–X coordination pattern from octahedral to trigonal prismatic.In 2 monolayers192 monolayers is very likely to happen under normal laboratory experimental conditions.According to transition-state theory, assuming a characteristic attempt frequency of 10 THz, which is the typical frequency of optical phonons in 1T′–MX2 monolayers is the formation of domain boundaries between different orientation variants. Strain-induced ferroelastic switching between the O1, O2 and O3 variants can lead to the formation of three possible types of coherent twin boundaries, between O1 and O2, O1 and O3, and between O2 and O3, which we refer to as O1–O2, O1–O3 and O2–O3, respectively. The DFT-relaxed atomistic structures of the three different types of twinning domain boundaries in 1T′–WTe2 monolayers under zero external stress are shown in 2 monolayers are quasi-one dimensional (1D) in nature, which may impart them unique properties. We have calculated the domain boundary energies associated with the three types of the 1D domain boundaries and found they have small formation energies. Our DFT calculations give the domain boundary energies of MoS2, MoSe2, MoTe2, WS2, WSe2 and WTe2 monolayers to be 27, 46, 40, 22, 51 and 52 meV· Å−1, respectively. In comparison, the formation energy of another type of 1D defects in 3D crystals, dislocations, is in the order of several hundred meV per Angstrom. Such small-domain boundary energies will facilitate the ferroelastic switching between different orientation variants.A direct consequence of strain-induced variant switching in 1T′–MX2 monolayers, with a few percent of local strain. Our calculations indicate that variant switching can most easily happen when stretching the 1T′–MX2 monolayers along the direction of dimerized metal chains. This prediction, if experimentally realized, will render 1T′–MX2 monolayers as the first class of 2D ferroelastic materialsin situ transmission electron microscopy experiments of mechanical deformation of 1T′–MX2 monolayers. As the domains of different variants have different crystallographic orientations, the migration of domain walls during strain-induced variant switching can be observed by dark-field transmission electron microscopy, which has been demonstrated for domain imagining in graphene and MoS2 monolayers33The thermodynamic and kinetic analysis above have provided strong evidence that strain-induced ferroelastic switching of orientation variants can occur in 1T′–MX2 and 1T′–MoTe2, for which bulk single crystals have been synthesized on a large scale and exfoliated down to the monolayer or few-layer regime202 few layers in 1T′ phase have been grown via chemical vapour deposition232 from the 2H to 1T′ phase can also be realized using laser ablationOur prediction of ferroelasticity in the TMD monolayers can be readily tested experimentally in 1T′–WTe2 that include MoS2, MoSe2, WS2 and WSe2, as the 2H phase is energetically more stable than the 1T′ phase under normal conditions, the 1T′ phase can be realized using a phase engineering approach372, MoS2 and MoSe2 in 1T′ phase have been obtained via liquid-phase exfoliation of the bulk crystals intercalated by alkaline metals18252 and 1T′–MX2 monolayers, and it is indeed shown that, with increased amount of adsorbed lithium, the 1T′ phase becomes energetically more favourable than the 2H phase for all the MX2 monolayers, as illustrated in 2 with elements having more valence electrons  than the transition metal ions can be another effective way to stabilize the 1T′ phaseFor other group VI MX2 monolayers, if the 1T′ phase can be reversibly transformed to the 1T or 2H phase under external stimuli , then MX2 monolayers could be 2D shape memory materials, with operating principles similar to SMAs. As such, 1T′–MX2 monolayers can be used to make ultrathin actuators for applications in nanoscale-integrated electromechanical systems.From an application perspective, ferroelastic behaviours have close connection to the shape memory effect, which has been exploited to make actuators in a wide range of industries. In 3D, shape memory alloys (SMAs) is a well known and technologically important class of ferroelastic materials. SMAs can undergo a diffusionless martensitic phase transformation below a critical temperature, from the high-temperature austenite to the low-temperature martensite phase2 monolayers were predicted to be quantum spin Hall insulators2 monolayers, considering the rich family of 2D materials2, NbTe2 and TaTe2, which have low-symmetry distorted crystal structures similar to 1T′2 and ReSe2 monolayers has recently been reportedIn closing, we would like to make a few additional comments. First, since the 1T′–MXz direction). The length of the supercells along the z direction was chosen to be 20 Å. Brillouin zone integration employed a Gamma point centred m × n × 1 Monkhorst-Packk-point grid and a Gaussian smearing of 50 meV, where the numbers m and n were chosen such that the k-point sampling spacing is <0.1 Å−1 along the supercell reciprocal vectors in the x–y plane. The energy convergence thresholds for electronic and ionic relaxations were 10−6 and 10−5 eV, respectively. The maximum residual forces resulted from these convergence criteria are smaller than 5 × 10−3 eV Å−1. We provide the DFT-relaxed atomistic structures of the O1, O2 and O3 variants of 1T′–MX2 monolayers in the POSCAR format of Vienna Ab initio Simulation Package, as listed in DFT calculations were performed using the Vienna Ab initio Simulation Package with a plane-wave basis set45How to cite this article: Li, W. et al. Ferroelasticity and domain physics in two-dimensional ransition metal dichalcogenide monolayers. Nat. Commun. 7:10843 doi: 10.1038/ncomms10843 (2016).Supplementary Figures 1-5, Supplementary Tables 1-9 and Supplementary References.