However, UCNP synthesis typically requires high temperatures in combination with either high pressure reaction vessels or an inert atmosphere. Here, we report synthesis of α-phase NaYF4:Yb,Er,Mn UCNPs via the considerably more convenient PVP40-mediated route; a strategy that requires modest temperatures and relatively short reaction time  in open air, with Mn2+ co-doping serving to greatly enhance red emission. The optimal Mn2+ co-doping level was found to be 35 mol %, which decreased the average maximum UCNP Feret diameter from 42 ± 11 to 36 ± 15 nm; reduced the crystal lattice parameter, a, from 5.52 to 5.45 Å; and greatly enhanced UCNP red/green emission ratio in EtOH by a factor of 5.6. The PVP40 coating enabled dispersal in water and organic solvents and can be exploited for further surface modification (e.g. silica shell formation). We anticipate that this straightforward UCNP synthesis method for producing strongly red-emitting UCNPs will be particularly beneficial for deep tissue biophotonics and biosensing applications.Cubic (α-phase) NaYF These properties enable deep tissue biological imaging without visible autofluorescence from biological components such as tissue and blood, and without the phototoxicity associated with conventional high energy UV/visible excitation .,27.4, Nasamples) ,28. UCNPand Tm3+ . Core–shand Tm3+  and to mand Tm3+ ,31–34.FUCNP synthesis presents some challenges which can limit rate and scale of production. The most commonly used UCNP synthesis procedures, i.e. hydro/solvothermal and thermal decomposition methods, require either an autoclave reaction chamber capable of withstanding elevated pressures (e.g. approx. 3 MPa) , or othe3+ and Na+ ions, enabling formation of UCNPs via controlled precipitation when F− ions are introduced  (99.99%); ytterbium(III) oxide [Yb2O3] (99.9%); erbium oxide [Er2O3] (99.9%); PVP 40 000 (PVP40) [(C6H9NO)x]; ammonium fluoride [NH4F] (greater than 99.99%); and sodium chloride (greater than 99.5% BioXtra) were purchased from Merck Life Science UK Ltd. Manganese(II) nirate tetrahydrate [Mn(NO3)2 · 4H2O] (98%); 70% nitric acid (HNO3) (laboratory reagent grade); ethylene glycol (EG) [(CH2OH)2] (greater than 99% extra pure ACROS Organics); and absolute EtOH  were purchased from Fisher Scientific. All materials were used without further purification.Yttrium(III) oxide [Y. 2.22O3, Yb2O3, Er2O3 were prepared in 10% nitric acid. These required preparation several days in advance due to the poor solubility of these lanthanide oxides at room temperature. Elevated temperatures were not used to assist dissolution in order to avoid thermal decomposition of nitric acid (clear colour) to nitrogen dioxide (yellow colour). Stocks of Mn(NO3)2 · 4H2O were prepared as required. Stock solutions of NH4F and PVP40 in ethylene glycol were prepared at room temperature at least 24 h in advance, with the PVP40 stock solution requiring vigorous manual stirring with a glass rod for 5 min.The PVP40-mediated UCNP synthesis method used herein was adapted from prior studies ,48,49. F2O3, Yb2O3, Er2O3 and Mn(NO3)2·4H2O stocks in 10% HNO3 were combined in various amounts as per electronic supplementary material, table S1 to create a transparent solution containing a total of 1 mmol of Ln3+ and Mn2+ ions. This solution was then vigorously stirred for 1 min before heating at 120°C to evaporate the aqueous content, resulting in either a transparent residue (0 mol% Mn2+) or a brown residue (with Mn2+); the solution was removed from the heat when the residue stopped bubbling. A total of 8 ml of ethylene glycol was added, and the mixture was stirred at 80°C for a minimum of 30 min, or for as long as necessary for the solution to turn clear in colour. Once the solution was clear, 58.5 mg (1 mmol) of NaCl was added directly to the solution and stirred for 5 min at 80°C. Then 0.556 g/0.014 mmol of PVP40 stock (2 ml volume) was added dropwise and the mixture was stirred for 10 min at 80°C, after which the solution was transferred to a round bottom flask and maintained at 80°C via an oil bath. Mixture B was prepared by adding 8 ml of ethylene glycol to a conical flask and raising the temperature to 80°C. Then 4 mmol/148.2 mg of NH4F stock (2 ml volume) was added and the solution was stirred vigorously for 10 min. Mixture B was then added dropwise to Mixture A dropwise and vigorously stirred at 80°C for 10 min. The resultant solution was heated to 160°C via an oil bath at a rate of approximately 5°C min−1 and maintained at 160 ± 5°C for 2 h with vigorous stirring. As the reaction progressed, the solution clearly changed from semi-opaque and colourless to an opaque yellow/orange coloration, typically producing approximately 130 mg of PVP40-coated α-phase UCNPs. The UCNPs were collected by centrifugation at 10 000 relative centrifugal force (RCF) for 45 min. The supernatant was removed and replaced with 10 ml of EtOH. The UCNPs were then resuspended by sonication for 15 min. Two more wash steps were conducted (with centrifugation at 7000 RCF for 30 min), with final suspension of UCNPs in 5 ml of EtOH.UCNPs were prepared as follows. Mixture A: Y. 2.32 X-max X-ray detector for EDS measurements. UCNP diameter measurements were conducted manually with FIJI and tabulated in Microsoft Excel [i) were analysed with a minimum of 100 UCNPs in each sample. For scanning electron microscopy (SEM), UCNP samples were prepared in a similar manner to TEM samples and 10 µl of each sample was gently dropped onto a pre-washed silicon wafer (Agar Scientific) and heated at 50°C using a hotplate to evaporate the isopropyl alcohol. Each silicon wafer was then mounted onto a stub mount and imaged with an SEM  using the InLens detector at 10 000×, 30 000× and 100 000× magnifications.Transmission electron microscopy (TEM), electron dispersive spectroscopy (EDS) and selected area electron diffraction (SAED) were conducted as follows. Samples were prepared for imaging by sonicating the as-prepared UCNP solutions for 15 min and then adding 10 µl of UCNP sample to approximately 1 ml of isopropyl alcohol. This was then sonicated for 2 min, and a single drop was placed onto a holey carbon-supported copper grid and allowed to dry at room temperature for 24 h. TEM imaging was conducted on JEOL JEM-2100F operated at 200 kV with an Oxford Instruments 65 mmft Excel . The minkα source). The precise two-theta angle of each XRD peak was found by fitting a Gaussian profile to each peak using a custom-written analysis script . The cubic (α-phase) crystal lattice parameter, a, was calculated from the average estimate from eight major via the following equations:hkld is the interplanar spacing, λ is the X-ray wavelength, 2θ is the two-theta angle of each peak and hkl are the Miller indices corresponding to each peak.Dry powder UCNP samples  were prepared for X-ray diffraction (XRD) and inductively coupled plasma mass spectroscopy (ICP-MS) measurements as follows. Approximately 10 mg of as-prepared UCNPs in EtOH was transferred to an open glass vial inside a fume hood and the EtOH was allowed to evaporate naturally. The resultant dry residue was then scraped within the vial to produce a loose white powder. For XRD measurements, the powdered sample was placed onto a silicon slide and held in place with a binding agent (Vaseline). Scans were conducted using a Bruker AXS D8 Advance GX 003 410 fitted with a Lynxeye Soller PSD detector and automated sample changer, and operated in Bragg–Brentano mode with a Ni filter. XRD patterns were acquired between 10 and 90° in 0.02° steps, with variable slit width fixed at 6 mm, and using a wavelength of 1.5406 Å  and inductively coupled plasma mass spectroscopy (ICP-MS). For these measurements a known mass of dry powdered sample was transferred to a perfluoroalkoxy alkane (PFA) vial. Then 3 ml of aqua regia was added, the vial sealed and heated to 100°C for 12 h. The sample was then diluted to 50 ml on the day of analysis. ICP-AES measurements were conducted using a Thermo Scientific SICAP 6000 previously optimized for sensitivity and signal stability. Multiple emission lines were measured for each element to monitor any spectral interference that may be present. ICP-MS measurements were conducted using a Thermo Scientific X-Series.−1. The cuvette was placed inside an enclosed light-proof sample chamber for measurement. Excitation was provided by a femtosecond pulsed tuneable NIR laser  with a stated spot size of 1.2 mm and variable power output, with a typical maximum power of 1300 mW at 980 nm. The laser was routed to the sample chamber via beam routing mirrors (Thorlabs BB1-EO3) within light-tight tubes. The laser beam was focused onto the sample by an ultrafast 50 mm laser lens  resulting in an approximate beam spot size of approximately 62 µm. Luminescence was measured 90° to excitation by an Ocean Optics HR2000Pro spectrometer . For standard luminescence intensity measurements, the excitation laser power was held at 70%, i.e. approximately 960 mW at 980 nm; 1000 spectra were acquired and averaged, with each spectrum being a 50 ms acquisition . UCNP samples were shaken gently before and after each measurement to ensure UCNPs were well suspended. The spectrometer was operated using a custom-written LabVIEW program (LabVIEW 2013). Emission was filtered by a less than 700 nm short-pass filter  to ensure no stray excitation light reached the detector. Luminescence was analysed by custom Matlab scripts. Green emission intensity was quantified by the total emission area under the curve arising from the 2H11/2 → 4I15/2 (approx. 521 nm) and 4S3/2 → 4I15/2 (approx. 545 nm) emission transitions; red emission intensity was quantified as the total area under the emission curve of the 4F9/2 → 4I15/2 transition (approx. 660 nm). For measurements of UCNP luminescence versus excitation power, the UCNPs were suspended in ethylene glycol to avoid gradual sedimentation of UCNPs.For luminescence measurements, 5.4 mg of as-prepared samples of UCNPs in EtOH were added to a 1 cm path length quartz cuvette  and the volume was adjusted to 0.9 ml by adding EtOH for a final UCNP concentration of 6 mg ml. 3a–g, with corresponding diameter distributions shown in figure 3j,k. UCNP diameter and aspect ratios are summarized in 4:Yb,Er  UCNPs produced (figure 3a) are very similar in form and diameter (maximum Feret diameter = 42 ± 11 nm) to UCNPs synthesized previously using the same method by Grüner et al. (diameter approx. 45 nm) [2+ resulted in an increase of UCNP diameter to 60 ± 19 nm, but no change in overall UCNP aspect ratio. Similar results were obtained with 20 mol% Mn2+ co-doping. Notably, a Gaussian diameter distribution was observed for 0, 10 and 20 mol% Mn2+ samples. Increasing Mn2+ to 25 mol% produced UCNPs of a considerably smaller diameter (maximum Feret diameter = 43 ± 16 nm) and slightly lower aspect ratio. Notably, the diameter distribution of these UCNPs was lognormal instead of Gaussian. This trend continued for 30, 35 and 40 mol% Mn2+ , which produced smaller UCNPs . These smaller UCNPs were also less homogeneous in form  , indicat in form . SEM imaurements .Table 12+ UCNPs attributable to α-phase NaYF4 via comparison with test card data . Acquisition of SAED measurements was attempted for 35 mol% Mn2+ UCNPs, but crystal planes suitable for analysis could not be identified. From powder XRD measurements, the α-phase crystal lattice parameter, a, was calculated to be a = 5.521 ± 0.003 Å for NaYF4:Yb,Er  UCNPs. This value is identical to the value reported by Sikora et al. (5.52 Å) for UCNPs of the same composition synthesized with the same method [Powder XRD pattern analysis confirmed that UCNPs produced were cubic α-phase, with all lines produced by 0, 10 and 20 mol% Mnard data . SAED fre method , indicat2+ mol% decreased the crystal lattice parameter, a, corresponding to a decrease in overall unit cell volume. The minimal value was a = 5.448 ± 0.002 Å for 35 mol% Mn2+ UCNPs (i.e. a 1.3% decrease from 0 mol% Mn2+ UCNPs). Intriguingly, a increased to a = 5.470 ± 0.007 Å for 40 mol% Mn2+ UCNPs. This trend was reflected in a shift towards higher 2θ angles for all samples between 0 and 35 mol% Mn2+, with a similar reversal for 40% Mn2+ . Similar shifts in 2θ angle have been reported for upconversion materials co-doped with Fe3+ and Mn2+ [Increasing Mnand Mn2+ ,57–60.2+ co-doping greater than or equal to 25 mol% (θ = ∼20° and ∼39.5° may be attributable to (100) and (111) XRD peaks of β-phase NaYF4. However, the many other β-phase NaYF4 peaks did not arise. Further, a peak at 2θ = ∼26.7° arises which was not attributable to β-phase NaYF4 . Therefore, it is unclear as to exactly what crystal phase is formed at Mn2+ co-doping levels greater than or equal to 25 mol%. Other studies have postulated that incorporating Mn2+ into upconversion lattices otherwise occupied by Ln3+ can result in formation of F− vacancies, which will alter the crystal lattice structure and induce lattice contraction, consistent with XRD observations [2+ co-doping levels greater than or equal to 25 mol%. However, a candidate structure may be orthorhombic phase NaMn3F10 as previously inferred by Wang et al. [Three new minor XRD peaks were observed in UCNPs samples of Mnrvations . With thg et al. .2+ co-doping enhanced red emission of the UCNPs . The red/green emission ratio of samples had a clear anti-correlation with the UCNP lattice parameter, a . Notably, there was negligible emission from the 2H9/2 → 4I15/2 emission transition (λem = ∼410 nm), indicating that this higher energy transmission was not sensitized in any of the UCNPs produced.Mnhe UCNPs . For 0 md method ,48,49, a2+ and 35 mol% Mn2+ UCNP in ethylene glycol  shows a two-photon dependence for 0 mol% Mn2+ UCNPs (m = 1.51), and a three-photon dependence for 35 mol% Mn2+ UCNPs (m = 2.49). This is consistent with the proposed upconversion process depicted in 4F9/2 → 4I15/2 Er3+ transition is populated by energy transfer from 4T1 energy level of Mn2+ ions, which is in turn populated by the 4F9/2 energy level of Er3+ ions, and which is populated in turn by multi-photon upconversion from Yb3+ donors. This would explain why even modest Mn2+ co-doping resulted in increased red emission. Notably, emission via this transition was lessened for 40 mol% Mn2+ UCNPs, which may be indicative of non-radiative energy loss processes such as energy transfer between adjacent Mn2+ ions. The 2H11/2 → 4I15/2 and 3S3/2 → 4I15/2 emission pathways (λem = ∼521 and 545 nm respectively) demonstrate a three-photon power dependence for both 0 mol% Mn2+ and 35 mol% Mn2+ UCNPs. The UCNPs samples saturated at laser powers greater than 5% of maximum laser power, which due to the focused excitation beam, corresponded to an approximate power at the focal spot of 2150 W cm−2. This is similar to saturation processes observed for other upconversion materials [The multi-photon upconversion process was investigated by varying the excitation laser power and monitoring the emission of 0 mol% Mne glycol . In unsach plots . The mosaterials –63.Figu2+ co-doping. Prior studies have shown that Mn2+ co-doping can dramatically improve red emission in a variety of UCNP morphologies produced via solvothermal and hydrothermal methods [2+ co-doping encourages formation of pure α-phase UCNP structure, which is well suited for red emission due to larger unit cell size than the smaller unit cells of β-phase UCNPs [2+ co-doping resulting in suppression of the 4S3/2 → 4I15/2 (green) emission transition entirely in favour of the 4F9/2 → 4I15/2 (red) emission transition [2+ co-doping dramatically increases red emission in α-phase UCNPs produced via the relatively convenient PVP40 route without inducing an overall change in crystal phase. While pure single-band red emission was not achieved, multi-band red and green emission can be useful, e.g. for ratiometric sensing applications. The relative simplicity of the PVP40-mediated UCNP synthesis method makes it an attractive means of producing UCNPs without rate limitation requirements of hydrothermal/solvothermal pressure vessels. Yet the wider potential of the PVP40-mediated UCNP synthesis route has only barely been explored in publications to date [Here we have shown the PVP40-mediated UCNP synthesis method can be used to create UCNPs with strong red emission via Mn methods ,40,50–55ansition ,65. Wher to date ,66,67. T3+ ions within the host lattice in favour of Yb3+ sensitizer ions has been shown to considerably enhance the red emission of cubic NaYbF4:Er,Mn UCNPs synthesized via solvothermal methods [et al. indicate that increasing Yb3+ cation dopant concentration may improve population of the 4F9/2 → 4I15/2 emissive pathway for NaYF4:Yb,Er UCNPs synthesized via the PVP40 route [3+ instead of Er3+ as the emissive ion [3+ co-doping could also enhance red emission in α-phase UCNPs [3+, Gd3+, Cu2+, Pr3+ and Ho3+ may also be of interest [3+ being particularly notable for their use as dual-mode reporters for both optical luminescence and magnetic resonance imaging (MRI) [For example, completely forgoing the photodynamically inactive Y methods . Indeed,40 route ,49. Bluesive ion . Fe3+ cose UCNPs . Other cinterest ,59,68, wng (MRI) . While tng (MRI) .3)3 · 6H2O, Yb(NO3)3 · 5H2O and Er(NO3)3 · 5H2O), which have excellent solubility in 10% nitric acid. Despite otherwise identical reaction conditions, these starting materials formed in inhomogeneous pseudo-nanorods with no apparent photonic upconversion activity . ICP-MS results indicated that Na+ was not present in these samples, indicating a disrupted UCNP synthesis for unclear reasons, presumably due to the lanthanide nitrate starting materials. More promisingly, rare earth chlorates have been used as starting materials for PVP-mediated synthesis of green-emitting UCNPs for solar-cell applications [The use of PVP for controlled synthesis of UCNPs could enable further tuning of UCNP properties by varying the molecular weight/chain length of the PVP used. For example, in seed-mediated Ag nanoparticle synthesis, the molecular weight/length of PVP chain used can dramatically alter the size and shape of nanoparticles produced . It is aications –72. Prioications ,44,48.. 44:Yb,Er,Mn UCNPs with strong red emission can be produced in a convenient manner via the PVP40-mediated synthesis route. This method requires only simple hot plate and beaker apparatus and can be conducted under standard atmosphere at relatively low temperatures of 160°C with a relatively short 2 h primary reaction time. Mn2+ co-doping also altered unit cell composition with currently unidentified crystal lattice structures appearing at Mn2+ co-doping levels of greater than 25 mol%. Crucially, Mn2+ co-doping resulted in increased sensitization of the 4F9/2 → 4I15/2 Er optical emission pathway, with 35 mol% Mn2+ co-doping maximizing red emission, minimizing the unit cell size and producing overall smaller UCNPs with modified morphology and reduced aspect ratio. The UCNPs produced by this method are probably well suited to biomedical applications due to their strong red emission and average maximum Feret diameter of 36 ± 15 nm.We have demonstrated that PVP40-coated α-phase NaYF


Pseudomonas balearica RAD-17 in the presence of ciprofloxacin (CFX) and oxytetracycline (OTC). No significant negative impact on the performance of aerobic denitrification in the presence of CFX or OTC within the range of 50 to 300 μg L−1 was found. Significant degradation of OTC was found within the range of 50 μg L−1 to 300 μg L−1 under aerobic denitrification conditions, while no degradation was found for CFX. Stimulation of cell growth occurred within the investigated range of antibiotics. Under anoxic or aerobic conditions, the addition of CFX or OTC changed the N2O production trend. The results in the present study may play an important role in informing the use of aerobic denitrification techniques in the presence of antibiotics within environmentally relevant concentrations (<1 mg/L).Aerobic denitrification, one of the important nitrate metabolic pathways in biological denitrification, has been attracting increasing interest recently due to its functional advantages. In order to evaluate the effect of antibiotics on aerobic denitrification and guide practical engineering application of aerobic denitrification techniques, we evaluated the performance of aerobic denitrification by the strain  In the  microbe . As a reAntibiotic overuse, however, is now a common phenomenon and has led to myriad problems . For insnirK and nosZ can be inhibited by TCs at low concentrations (10−3 mg L−1) [2O was found during denitrification process due to the inhibition of nosZ by TCs. In the past, more attention was paid for the effect of antibiotics on anoxic denitrification process. Effect of antibiotics on the performance of aerobic denitrification by pure strain has not gained enough attention.Antibiotics are one of the most complex factors impacting the denitrification process. effects of antibiotics (such as TCs) on denitrification processes have attracted scientific attention. Research on the effect of TCs on denitrification is very limited and has mainly focused on the matrices of bioreactors ,18,19,20 mg L−1) . As a rePseudomonas balearica RAD-17, isolated by us from a PBS (poly-butylene succinate) based denitrification reactor used for aquaculture effluent treatment, was employed to investigate the performance of aerobic denitrification of RAD-17 in the presence of ciprofloxacin (CFX) and OTC. The purpose of this research was (1) to evaluate the performance of aerobic denitrification with CFX or OTC; (2) to investigate the growth behavior of RAD-17 and the effect of CFX or OTC on the process of aerobic nitrogen; and (3) to elucidate the mechanism of interaction between nitrate and the strain RAD-17, and then guide practical engineering applications of aerobic denitrification technique.In the present research, the strain Pseudomonas balearica , based on 16S rRNA phylogenetic analysis with the Accession Number NCB: MK881511. The strain RAD-17 is a gram-negative bacterium that is 0.3–0.4 μm in diameter and 0.8–1.6 μm in length. In addition, according to the complete genome sequence results (NCBI: PRJNA599547), gene annotation, and KEGG pathway results, the strain RAD-17 embodies a series of genes for denitrification under aerobic conditions. Under aerobic conditions, nitrate-nitrogen (NO3−–N) and nitrite nitrogen (NO2−–N) can be removed at rates of 6.22 mg L−1 h−1 and 6.30 mg L−1 h−1, respectively [−1 h−1 [The strain RAD-17, employed in the current research, was isolated from a PBS (poly-butylene succinate) based denitrification reactor for the treatment of aquaculture effluent. The operating conditions for the reactor can be found in , and infectively . Further [−1 h−1 .v/v) was inoculated into denitrification media (DM) for nitrogen removal evaluation in 250 mL Erlenmeyer flasks (100 mL solution). The DM was prepared at the ratio of KNO3 , sodium acetate , 0.2 g/L MgSO4·7H2O, 1.0 g/L K2HPO4, and 1.0% (v/v) trace-element solution. The trace-element solution contained 50.0 g/L ethylenediaminetetraacetic acid (EDTA), 2.2 g/L ZnSO4, 5.5 g/L CaCl2, 5.06 g/LMnCl2·4H2O, 5.0 g/L FeSO4·7H2O, 1.1 g/L (NH4)6Mo7O2·4H2O, 1.57 g/L CuSO4·5H2O, 25 g/L NaCl and 1.61 g/LCoCl2·6H2O [−1. To exclude abiotic degradation, no inoculation was carried out for the DM containing 300 μg L−1 CFX or OTC and served as the abiotic degradation control. Before and after 48 h, under incubation conditions of 150 rpm at 30 °C, the concentration of total organic carbon (TOC), NO3−-N, NO2−-N, NH4+-N, and CFX or OTC were determined. There were three replicates for this experiment. Moreover, dynamic degradation experiments of CFX or OTC were also carried out with the highest antibiotic concentrations (300 μg L−1). During this experiment, the concentration of CFX or OTC at 12, 24, and 48 h was determined, and five replicates for each treatment were employed. Detailed information on the CFX or OTC analysis procedures can be found in our recently published reports [To evaluate the performance of aerobic denitrification for strain RAD-17 with co-existing antibiotics, a batch experiment was carried out under laboratory conditions; this is described here briefly. First, the seed solution was prepared and incubated for 48 h. Then, a seed solution of 1% (v/v), which contained 0 μg L−1 and 300 μg L−1 of CFX or OTC before being incubated under aerobic conditions. The strain solutions were sampled at 12 h, 24 h, and 48 h and were diluted with sterile saline solution  to reach a cell density of 1.0 × 106 cells ml−1 to 1.0 × 107 cells ml−1. Afterward, 0.5 mL of diluted strain solution was sampled, and then the determination was carried out by flow cytometry . Detailed information on the calculation of the strain cell density can be found in our recent publications [To evaluate the variation of strain cell numbers, a flow cytometry analysis experiment was carried out. After pre-culturing in DM, the strain RAD-17 was inoculated into new DMs . After incubation for 48 h at 30 °C under aerobic or anaerobic conditions, 5 mL of the headspace product was sampled using a 25 mL syringe for N2O analysis by gas chromatography . Detailed information on the analysis procedure can be found in our recently published reports [After pre-culturing in DM for 48 h at 30 °C, 0.5 mL of strain solution was inoculated into 50 mL medium in 100 mL headspace vials (with rubber stoppers), which contained 0 μg L3−-N, NO2−-N, and NH4+-N followed the standard methods [600) were determined by a TOC analyzer  and spectrophotometer at 600 nm, respectively. In addition, the inhibition rate of the net OD600 value was also calculated by the following formula:Samples for chemical analysis were first filtered through a 0.45 μm filter membrane, and then the determination of NO methods . TOC andThe statistical analysis was performed using SPSS 20.0 . In order to evaluate the difference between different treatments, a one-way analysis of variance was employed.−1 to 300 μg L−1 (3−-N L−1 h−1) in the presence of 200 μg L−1 was significantly higher than all the other treatments. In the presence of OTC, there were also no significant impacts on the aerobic denitrification performance of strain RAD-17 within the range of 50 μg L−1 to 300 μg L−1 , respectively. However, the net OD600 value varied from 0.71 ± 0.16 to 1.02 ± 0.34 with the addition of 50 to 300 μg L−1 of CFX. Accordingly, the net OD600 value and growth yield varied from 2.30 ± 0.40 × 10−3 OD600/(mg NO3−-N) to 4.10 ± 0.13 × 10−3 OD600/(mg NO3−-N). In the presence of OTC, a more serious impact on the growth of strain RAD-17 was found than in the presence of CFX , respectively. In addition, the net OD600 value varied from 0.53 ± 0.10 to 0.73 ± 0.11 with the addition of 50 to 300 μg L−1 of OTC. Accordingly, the net OD600 value and growth yield varied from 2.10 ± 0.30 × 10−3 OD600/(mg NO3−-N) to 2.70 ± 0.70 × 10−3 OD600/(mg NO3−-N).In order to evaluate the impact of antibiotics on the behavior of strain RAD-17, the changes of the ODn RAD-17 A. Withoue of CFX B. The ne600 value and cell numbers were also investigated with the addition of CFX and OTC. Without antibiotic treatment, no significant change was found in a 12 h to 48 h cultivation period for the net OD600 value, which varied from 1.24 ± 0.03 to 1.33 ± 0.26  was determined to evaluate the effect on the denitrification process of CFX and OTC, and different trends were found among the three treatments (2O content under anoxic conditions (1.38 ± 0.80 ppm) was significantly higher than that under aerobic conditions (0.30 ± 0.25 ppm) (p = 0.05). The opposite trend was observed in the presence of CFX; the N2O content under aerobic conditions (2.05 ± 0.57 ppm) was significantly higher than that under anoxic conditions (0.87 ± 0.19 ppm) (p = 0.05). However, no significant change was found for the N2O content between aerobic conditions (1.44 ± 2.22 ppm) and anoxic conditions (1.49 ± 0.80 ppm).Nitrous oxide  (p = 0.05). The nitrate removal rates of RAD-17 were in accordance with previous reports on pure strains, with or without addition of antibiotics, e.g., 6.47 mg NO3−-N L−1 h−1 (without antibiotics) and 5.72 to 6.46 mg NO3−-N L−1 h−1 (in the presence of antibiotics) for strain RAD-2 [3−-N L−1 h−1 for strain RAD-17 (without antibiotics) [−1) and decreased significantly under high level OTC stress (5 mg L−1) [−1 TC [In the current research, no significant impacts on the aerobic denitrification performance of strain RAD-17 were found within the range of 50 μg Libiotics . Furtherbiotics) . As ment mg L−1) . Similar) [−1 TC .Pleurotus ostreatus mycelium was found to remove Oxytetracycline (OTC) completely within 14 days [Trichoderma deliquescens RA114, Penicilium crustosum RA118 [Fusarium sp. CMB-MF017, and Paecilomyces sp. CMBMF010 [−1) were degraded by thestrain Sphingobacterium sp. PM2-P1-29, which contains the tet (X) gene [Stenotrophomonas strain was isolated from soil, and has been shown to capable of degrading 44.43 mg L−1 of TCs within four days [In the current research, there were high removal efficiency for OTC under aerobic denitrification process. Physical and chemical processes are commonly used abiotic techniques for the removal of TCs, and include filtration, adsorption, and oxidation ,6,7. Amo 14 days . In addi 14 days , Trichodum RA118 , FusariuCMBMF010 . In comp(X) gene . In addiour days .napA, nirs, norB, and nosZ) and the direct receptor genes should be investigated, and this is useful to understand aerobic denitrification behavior. For instance, different concentration of N2O were found under aerobic and anoxic conditions, and this might be related to expression of nosZ. (2) Cocktail effects of antibiotics to aerobic denitrification. In the current and some other related studies, single type of antibiotics was employed for the effect of antibiotics on aerobic denitrification. In a more realistic situation, multiple types of antibiotics rather than single type antibiocis were found. Thus, synergy or antagonism effect among multiple types of antibiotics should be also investigated in future. In the current research, high removal efficiency for aerobic denitrification were achieved in the presence of CFX or OTC. Furthermore, simultaneous aerobic denitrification and OTC degradation was found. However, before application of aerobic denitrification techniques in the presence of antibiotics, satisfied solution needs to be searched for the following issues: (1) aerobic denitrification pathways should be clearly understood. In the presence of antibiotics, expression of denitrifying genes (e.g., −1 to 300 μg L−1 under aerobic denitrification conditions, while no degradation was found for CFX. Stimulation of cell growth might occur within the investigated range of antibiotics. Under anoxic or aerobic conditions, the addition of CFX or OTC changed the N2O production trend. In the future, it is necessary to investigate the effect of antibiotics on the metabolic pathway by metagenome and macrotranscriptome methods and find the crucial genes involved in the denitrification-related pathway.There was no significant influence on the aerobic nitrate removal efficiency of strain RAD-17 with simultaneous denitrification and antibiotic degradation, regardless of the antibiotic concentration. Significant degradation of OTC was found within the range of 50 μg L


This compound simultaneously exhibited metallic conductivity and SMM behaviour up to 11 K for the first time. The highest electrical conductivity was 400–650 S cm−1 at 6.5 K, which is the highest among those reported so far for conducting SMM materials. Furthermore, antiferromagnetic ordering occurred below 6.5 K, along with a decrease in conductivity, and the angle-independent negative magnetoresistance suggested an effective electron correlation between the conducting BO and Co(pdms)2 SMM layers (d–π interactions). The strong magnetic anisotropy and two-dimensional conducting plane play key roles in the low-temperature antiferromagnetic semiconducting state. BO4 is the first compound exhibiting antiferromagnetic ordering among SMMs mediated by π-electrons, demonstrating the synergetic effects between SMMs and molecular conductors.Single-molecule magnets (SMMs) show superparamagnetic behaviour below blocking temperature at the molecular scale, so they exhibit large magnetic density compared to the conventional magnets. Combining SMMs and molecular conductors in one compound will bring about new physical phenomena, however, the synergetic effects between them still remain unexplored. Here we present a layered molecule-based compound, β′′-(BEDO-TTF) A metallic single-molecule magnet was synthesised demonstrating simultaneous metallic conduction and excellent SMM properties at the same temperature range for the first time, with potential applications in molecule-based quantum spintronics. Day and H. Kobayashi et al.2 The discovery of the giant magnetoresistance (GMR) effect by A. Fert et al.3 and P. Grünberg et al.4 in 1988 established the area of spintronics and significantly improved the size and storage capacity of magnetic digital data drives in the 21st century. The research in this field has concentrated on combining molecular conductors, such as bis(ethylenedithio)tetrathiafulvalene (BEDT-TTF),5 7,7,8,8-tetracyanoquinodimethane (TCNQ)6 and various single-molecule magnets (SMMs), such as [Mn2III2]− and [Dy(CF3COO)4]−,5,7 where 5-MeOsaltmen is the N,N′--bis dianion. SMMs can magnetically store information at a molecular level.8,9 Thus, they are good candidates for use in molecular spintronic devices, like spin-valves, and due to their long relaxation times, they are applied in quantum computers.10 Moreover, the combination of SMMs and electrical conductors may allow for nanoscale functionality beyond Moore's limitation.11Exploitation of multifunctionalities on the nanoscale is a critical challenge in modern chemistry to satisfy the rapidly growing demands in electronics.12 however, so far obtained materials exhibited semiconductor behaviour and SMM behaviour in different temperature ranges. They conduct above liquid nitrogen temperature and behave as SMMs at liquid helium temperature, and no synergism has been observed. Therefore, the search for suitable molecular conductors and SMMs has been intensively pursued. Recently, our group has reported (TTF)2[Co(pdms)2], which behaves as a semiconductor and an SMM in the same temperature region.13 Due to its unusual mixed stacking structure, strong charge transfer between TTF and [Co(pdms)2] affords electrical conduction down to 2 K with SMM properties up to 5 K, however, it is not metallic.The construction of conducting SMMs has been examined;14 The difference between the two molecules lies in the fact that, in BO, the four sulfur atoms in the six-member rings of ET are substituted by smaller and lighter oxygen atoms in BO, which can effectively shorten the intermolecular S⋯S distance between the central TTFs. In crystals of metallic BO complexes, donor molecules aggregate into layered structures, forming uniform (such as I3-type) arrays connected by intrastack C–H⋯O hydrogen bonds and side-by-side heteroatom short contacts to form a stable 2D metal.15 Following this idea, (BEDO-TTF)4(ReF6)·3H2O was the first example of the application of BO molecules to preserve relatively high σ down to low temperature (σ4.2 K = 47 S cm−1) for which the field-induced slow relaxation of magnetization was observed below 4 K, but no synergetic effects were reported.16 On the other hand, we selected a four-coordinate Co-based SMM, [Co(pdms)2]2− ,13 which is necessary for high energy barrier SMMs. The low energy absorbance band at 1200 nm  corresponds to the electron transition from the 4B1 orbital to the 4E orbital of the cobalt ion 4[Co(pdms)2]·3H2O, which has the highest σ value  in the family of conducting SMM materials and exhibits SMM behaviour in the antiferromagnetic phase with a TB up to 11 K. The synergetic effect, which is expected from the band structure calculation, has been found as the antiferromagnetic interactions and magnetoresistance (MR) at low temperatures.The oxygen analogue of BEDT-TTF (ET) is BEDO-TTF (BO) , which idms)2]2− . 2− to give the charge neutrality of (BO)4[Co(pdms)2]·3H2O. bc plane. The SMM layer alternates with the metallic layer along the c-axis. The thicknesses of the layers are 7.2 Å and 11.0 Å, respectively, indicating that both of them are 2D nanosheets. Numbers of hydrogen-bonds (C–H⋯O) are observed between BO and SMM layers in addition to those within the BO layer, both of which stabilize the crystal structure  between three Co ions in one parallelogram is 42°. The magnetic easy axes of all Co(pdms)2 units are aligned along the b-axis to show a strong one-dimensional (1D) magnetic anisotropy . While the intermolecular S⋯S and S⋯O atomic contacts shorter than the sum of van-der-Waals radii are observed along the a-axis (between side-by-side BO pairs) and along the a–b direction, significant magnitudes of intermolecular orbital overlap  are manifested along the a-axis and along the 3a + 2b direction  corresponds to the intermolecular charge-transfer (CT) transition between partially oxidized BO molecules. The absorption band at around 12 000 cm−1 was assigned to the electronic transition within a BO radical cation (electronic transition from the second HOMO to HOMO).15 In EPR spectra, a single peak with g = 2.004 was observed at room temperature , relaxation rate (Γ) and dielectric constant (ε∞) are evaluated as 6331.3 cm−1, 3394.2 cm−1, and 3.48, respectively. The reflectance spectrum indicates a highly conducting nature.18 Raman spectroscopy was carried out to determine the charge on the BO molecules to understand the degree of CT in the BO layer. The room-temperature Raman band at around 1480 cm−1 observed to be a superposition of two bands (∼1477 cm−1 and ∼1483 cm−1) was attributed to the totally symmetric C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C vibration of the BO (v3(Ag) mode, Fig. S6dv3(Ag) mode depends on the charge on BO and can be used to determine the charge density (ρ) using the following equation: ρ = (1524.9 − ν3(obs) (cm−1))/109.0.19 The possibility of inhomogeneous charges on BO molecules is thus supposed; the calculated charges are +0.44 and +0.38, respectively.In solid-state UV-Vis-NIR absorption spectra of  Fig. S6a. The low Fig. S6b, confirmχT of BO4, as a function of temperature in a 0.1 T field measured in zero field cooling (ZFC) and field cooling (FC) modes. The observed χT value of 3.22 cm3 K mol−1 at 300 K is 0.08 cm3 K mol−1 larger than the value expected for an S = 3/2 ion with g = 2.59 in (HNEt3)2[Co(pdms)2],20 which indicates that most of the paramagnetic susceptibility comes from Co(pdms)2. Hence, the contribution from the BO layer is estimated only to be around 2.7 × 10−4 cm3 mol−1, which is consistent with the metallic nature of the conducting layer. This χ value agrees well with the χ–T plot of (BO)4[Zn(pdms)2]·3H2O showing almost temperature-independent χ with the value of 2.8 × 10−4 cm3 mol−1  and Weiss constants (θ) were estimated to be 1.93 cm3 K mol−1 and +10.2 K by using the Curie–Weiss law. Thus, the indirect ferromagnetic interactions below 10.2 K between Co ions mediated by the pdms ligand were assigned. A. A. Fraerman et al. have reported that the region with ferromagnetic long-range interaction of a magnetic dipole system in an orthorhombic lattice is stable.22 It has been shown that the ground state of the system depends on the rhombic angle (φ). If φ < 60°, the dipoles are ordered ferromagnetically and the φ value of 42° observed for BO4 is regarded to be adequate to manifest ferromagnetic interactions in a parallelogram lattice . After rising again, χT abruptly decreased upon cooling below 6.5 K to 2.68 and 2.33 cm3 K mol−1 in ZFC and FC modes, respectively, indicating that there are antiferromagnetic interactions. To model the static magnetic properties, the spin Hamiltonian , where Si = SCo = 3/2 and where DCo stands for the zero-field splitting parameter for a CoII ion, was employed. Parameters of g(Co) = 2.19, DCo = −67.3 cm−1, and zJ = 1.05 cm−1 under FC conditions and g(Co) = 2.10, DCo = −59.0 cm−1, and zJ = 1.17 cm−1 under ZFC conditions were obtained from fitting 2 units in the SMM layer (TN), defined as peaks of the ln(χT)–T−1 plot, slightly shifted from 7.0 to 11 K when the magnetic field was increased from 200 to 10 000 Oe  of SMMs up to 11 K and TN up to 6.5 K. To the best of our knowledge, this is the highest TB among those reported for four-coordinate Co2+ based SMMs. M–H curve) in the initial field sweep and the dM/dH curve measured at 2.5 K. A stepwise increase was observed, indicating the presence of the spin–flop transition in the field of H1 and H2 (H2 > H1). The spin–flop transitions were observed in an H1 of 430 Oe and an H2 of 4700 Oe at 2.5 K and an H1 of 480 Oe at 5 K  magnetic susceptibility measurements (describe below). Therefore, the strength of antiferromagnetic interactions (zJ′) was calculated to be 0.0074 cm−1 by using a mean-field approximation model, 2|zJ′|ST2 = gSTμBH1 with ST = 3 and g = 2.0, where an ST of 3 was the largest magnetic moment (two ferromagnetic Co(pdms)2 units) in the SMM layer. H2 could be assigned to the coupling between the SMM and BO layers. Magnetic hysteresis loops were observed up to 5 K with a remnant magnetization of 0.08 Nβ and a coherence field of 23 Oe at 2.5 K, indicating that BO4 shows SMM behaviour  and out-of-phase (χ′′) ac magnetic susceptibility, χ′′, at 0 Oe at various temperatures. Dual relaxation peaks in the range of 0.1–1000 Hz, with the first relaxation process at low frequency and the second relaxation process at high frequency. The first relaxation process corresponds to the reversal of the spins of the Co ions, and the second relaxation process is related to the antiferromagnetic interactions between two adjacent SMM layers. The second relaxation process was suppressed by an optimized 1500 Oe field , leaving a single-relaxation process. τ) extracted from χ′′ in fields of 0 Oe  is much longer than that for (HNEt3)2[Co(pdms)2] at the 0 Oe field because of the ferromagnetic and antiferromagnetic interactions. Quantum tunnelling of magnetization (QTM) below 4 K was dominant, but it became suppressed at a frequency lower than that for the starting material due to the presence of weak antiferromagnetic interactions (H1). The energy barrier (Ueff) was determined by using the relaxation process at an optimized magnetic field. By considering the direct, Raman and Orbach processes for the fitting give no reasonable results. Thus, we consider here Orbach and Raman processes by using τ is best fitted using the following parameters: C = 1.42 × 10−5 s−1 Km−, m = 6.37, τ0 = 2.34 × 10−4 s, and Ueff = 29.21 cm−1. Τ0 for BO4 is almost six orders of magnitude lower than that for (HNEt3)2[Co(pdms)2] (τ0 = 1.31 × 10−10 s).20 Although the magnetic properties of BO4 are completely different from those of the starting material and Ueff is smaller than that of (HNEt3)2[Co(pdms)2], Ueff is similar to that of our recently reported conducting SMM (TTF)2[Co(pdms)2] .13 One possibility is that the D value is −67 cm−1, whereas for (HNEt3)2[Co(pdms)2], it is −115 cm−1.20 Fig. S12. This ma Fig. S11 and 1500 Fig. S11. The τfoσ at various pressures based on six single-crystals. At ambient pressure, σ (=50–110 S cm−1 at 295 K) increased with a decrease in temperature, behaving as a metal above 62 K. Then σ decreased down to 32 K, then increased again up to σ = 400–650 S cm−1 at 6.5 K, and finally drastically decreased to σ = 560 S cm−1 at 2 K. The change ratio σ(2 K)/σ(300 K) was 5–10. The decrease in σ at ∼62 K was completely suppressed by coating the crystal with Apiezon N grease, which is known to induce a weak pressure effect of ∼0.03 GPa on the crystal. However, the decrease in σ at low temperature could not be suppressed even when pressures up to 2.0 GPa were applied. To investigate the origin of the anomaly at low temperature under σ, the magnetic field was swept between −9 T and 9 T. The magnetic field B was oriented perpendicular to the current direction with an angle θ between the magnetic field and conducting plane (ab plane) adjusted by the rotator probe  − ρ]/ρ × 100%, where ρ is the resistivity. θ = 0° in the range of 2–100 K with symmetrical evolutions with respect to the field direction. The linear positive MR with 24% resistance increases at 2 K, and that with 2% resistance increases at 100 K in the field of 9 T. A negative MR was observed below 7.5 K in the low magnetic field region (θ = 0°) was caused by weak 2D localization or the interactions with the magnetic layer, the angle dependence of MR was examined. θ = 90° in the range of 2–100 K. The linear positive MR with 11% resistance increases at 2 K and that with 0.7% resistance increased at 100 K under a 9 T field. Furthermore, a negative MR was observed below 5 K in the low magnetic field region (θ = 90°) should have no effect on 2D localized electrons,23 however, a negative MR was also observed at 90° in contrast to the 2D localization model. This fact indicates that the decrease in σ observed below 6.5 K cannot be attributed to 2D weak localization. There must be interactions between the conducting and SMM layers in this temperature range.d region . In orded region . The negd region . A paral2ReO4·H2O.24 The bands of BO4 near the Fermi level mainly consist of eight HOMOs of the BO units, which are largely dispersed due to the effective HOMO overlap of the BO pairs in the “side” and “diagonal” directions . The Co(pdms)2 molecules are arranged in an ordered 2D narrow sheet (d = 7.2 Å), which causes the magnetic anisotropy of each Co(pdms)2 unit to be nearly aligned with the b-axis, affording a strong magnetic anisotropy. The ferromagnetic ordering observed between 10.2 K and 6.5 K is caused by weak intermolecular interactions between the Co(pdms)2 molecules due to the small rhombic angle in the magnetic layer. Subsequently, antiferromagnetic ordering was observed below 6.5 K assigned to the weak interactions between the SMM and conducting layers (d–π interactions). Thus, the weak inter- and intralayer interactions and the strong magnetic anisotropy maintain the SMM behaviour in a region of antiferromagnetic ordering. In other words, the Co(pdms)2 molecules are not significantly influenced by the surrounding Co ions, which essentially protects the SMM behaviour. On the other hand, we found that the TB of the Co(pdms)2 units significantly increased and the magnetic relaxation times increased below TN, which are caused by the interactions between conducting electrons and the SMM.The most striking physical property of σ at 62 K was suppressed by applying very low pressure (P = 0.03 GPa), which has been reported for several low dimensional organic metals,25 such as (BEDO-TTF)2ReO4·H2O and (TMTSF)2NO3, with a metal–insulator transition into a charge density wave (CDW) or spin density wave (SDW) state at low temperature.26 The σ value of (BEDO-TTF)2ReO4·H2O decreases several times due to the electronic instability involving the nesting of the hidden 1D Fermi surfaces (formed by the 2D hole and electron pockets).27BO4 has a similar arrangement of 2D Fermi surfaces of hole and electrons, and the decrease in σ at 62 K is ascribed to the formation of a CDW state due to the hidden 1D Fermi surface. The nesting of the 1D hidden Fermi surface along the ΓY direction  is enough to induce a change in σ.29For electron transport, the first decrease in σ below 6.5 K is insensitive to pressure. In this temperature range, a negative MR was observed for both perpendicular and parallel fields with respect to the conducting layer. Thus, the d–π interactions between the SMM and BO layers are important. Negative MRs were observed in fields perpendicular and parallel to the conducting layer up to ∼0.5 T, which is consistent with the characteristic spin–flop field (H2) in the MH curve. However, we should observe the corresponding distinct anomaly in the negative MR at the spin–flop transition (H2),30 and the absence of H2 in the MR measurement could be explained by the superposition of the largely positive MR enhanced by the peculiar semimetal-like electronic structure.The second decrease in 31 For the SMM layers, the TB of Co(pdms)2 was observed up to 11 K because of ferromagnetic coupling below 10.2 K. The increase in TB originates from the intrinsic ferromagnetic interactions. On the other hand, for the BO layers, the ab plane is metallic, and some electron density remains, although the partial nesting of the Fermi surface could occur. Thus, there is a possibility that the BO electrons are antiferromagnetically coupled with the SMM electrons along the b-axis below 6.5 K. Due to the two-dimensional conducting nature and strong magnetic anisotropy of the Co(d) electrons near the Fermi surfaces, synergetic effects could be observed. Furthermore, the presence of synergetic effects was supported by the low-temperature MR measurements where significant negative MR effects were observed in perpendicular and parallel directions.It is well known that a highly correlated electron system tends to adopt an antiferromagnetic insulating state.2]2− and neutral BEDO-TTF afforded β′-(BEDO-TTF)4 [Co(pdms)2]·3H2O (BO4), which showed the simultaneous manifestation of metallic conduction and SMM behaviours for the first time. Antiferromagnetic ordering mediated by π-electrons with SMMs was observed for the first time. By analysing the low-temperature magnetic phase transition and angle-dependence of magnetotransport, we found that the 2D conducting plane and strongly magnetic anisotropy played key roles in the synergetic effects in this layered material. From this aspect, SMMs have big advantages in molecule-based quantum spintronics compared to bulk magnets due to not only their high magnetic densities but also their strong magnetic anisotropies in SMMs. By combining them with high dimensional molecular conductors, such as two-dimensional BO and ET packing motifs, it is possible to observe such synergetic effects. Thus, from our studies on BO4, more stable metallic SMMs can be designed with the hope of producing superconducting SMM materials in the near future.Slow electrocrystallization of [Co(pdms)There are no conflicts to declare.SC-011-D0SC04040A-s001SC-011-D0SC04040A-s002


Clostridium sludge. Addition of 3.2 g/L exogenous butyric acid led to the highest 1.9 g/L butanol concentration with a conversion efficiency of 67%. With exogenous acetate and ethanol supply, the butanol concentration reached 1.6 g/L at the end of the incubation. However, the presence of acetic acid and ethanol favoured butanol production to 2.6 g/L from exogenous butyric acid by the enriched sludge. Finally, exogenous 14 g/L butyric acid yielded the highest butanol production of 3.4 g/L, which was also among the highest butanol concentration from CO/syngas fermentation reported so far. CO addition triggered butanol production from endogenous butyric acid  with as high as 58.6% conversion efficiency and 62.1% butanol yield. However, no efficient butanol production was found from glucose and CO co-fermentation (Glucose + CO), although a similar amount of endogenous butyric acid was produced compared to Glucose + N2. The Clostridium genus occupied a relative abundance as high as 82% from the initial inoculum, while the Clostridia and Bacilli classes were both enriched and dominated in Glucose + N2 and Glucose + CO incubations. This study shows that the supply of butyric acid is a possible strategy for enhancing butanol production by CO fed anaerobic sludge, either via exogenous butyric acid, or via endogenous production by sugar fermentation.Butanol is a potential renewable fuel. To increase the selectivity for butanol during CO fermentation, exogenous acetic acid and ethanol, exogenous butyric acid or endogenous butyric acid from glucose fermentation have been investigated using CO as reducing power, with a highly enriched  Thus, it can be used directly in any gasoline engine without modification and/or substitution, thus gaining more value than ethanol as a biofuel (Carbon monoxide (CO) is one of the main components in some industrial gas emissions such as steel plants and gasification of biomass or municipal solid waste . Syngas t decade . Butanol biofuel . CO ferm biofuel . In addiAcetobacterium woodii, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium carboxidivorans and Butyribacterium methylotrophicum, but very few can produce higher alcohols (Clostridium strains hardly produced butanol at concentrations higher than 2.7 g/L from syngas or CO (The conversion of CO follows the Wood–Ljungdahl pathway (WLP) to synthesize acetyl-CoA, that can be further converted to acetic acid, biomass and ethanol . Severalalcohols . Pure Clas or CO . Althougas or CO . Mixed cas or CO .Acid production in the acidogenic stage is necessary and enhances solvent production in the solventogenic stage . ExogenoClostridium kluyveri and solventogenic C. autoethanogenum  and co-fermentation of glucose and CO (Glucose + CO) by the enriched sludge.To date, selective butanol production from CO and exogenous acids and ethanol on butanol selectivity by mixed cultures has not yet been reported. This research addressed enhanced butanol production by enriched sludge with dominant populations of Clostridium genus reaching an ethanol production of as high as 11.8 g/L with only minor accumulation of acetic acid between pH 6.45 and 4.95. The inoculum could convert exogenous butyric acid into butanol using CO as the reducing power  was brought to boiling in order to remove O2, and it was later cooled down to room temperature under an oxygen-free N2 flow. Then CaCl2·2H2O and the vitamins were added, as well as Na2S (0.24 g) as reducing agent.The culture medium was prepared as described previously . A 1 L csolution . Once prAll experiments were carried out in 1 L fed batch reactors , with 10% enriched sludge in 200 ml medium. The fed batch reactors were sealed with a gas tight septum fitted with a pH probe  in the middle. The pH probe was connected to a pH controller  and the pH was adjusted using either 1 M NaOH or HCl solutions by two pumps . The fed batch reactors were agitated at 150 rpm by a shaker  at 33°C in a thermostatic chamber. 100% CO was supplied to the headspace of the reactor as the sole carbon and energy source with an initial gas pressure of 1.8 bar. When the gas pressure decreased below 1 bar, as a result of bacterial CO gas consumption (corresponding to one CO feeding), the reactor was flushed with fresh pure CO for about 5 min, until reaching again a gas pressure of 1.8 bar. Gas and liquid samples were taken once at each experimental time point and analyzed once.The effect of exogenous acetic acid, butyric acid and ethanol on the production of butanol by enriched sludge was evaluated in four 1 L fed batch reactors with 200 ml culture medium and inoculated with 10% enriched sludge . The medIn the HAc + EtOH experiment, acetate and ethanol addition was designed to stimulate the butyric acid production via the reverse β oxidation pathway and further induced butanol production using CO as reducing power. In the HAc + EtOH + HBu experiment, to demonstrate the enhancement of acetate and ethanol (reverse β oxidation pathway) on the butyric acid production, combined with exogenous butyric acid, which can possibly result in enhanced butanol production using CO as reducing power.An initial pH of 5.9 was applied and the pH was adjusted to 5.9 at each CO addition. The final concentrations of acetic acid, butyric acid and ethanol were, respectively, 2.2, 3.2 and 6 g/L . The aceCO was added at day 0, 11, 14, 15, 16 and 27 in the HAc + EtOH + HBu experiment; at 0, 11, 13, 15, 17, 20, 27 and 32 days in the HAc + EtOH experiment; at day 0, 17, 19, 20, 23, 25 and 30 in the HBu experiment; and at day 0, 20, 25, 27 and 35 in the control.Two 1 L CO fed batch reactors were used with 7 and 14 g/L butyric acid externally supplied at the same CO gas pressure and inoculum as described in 2 as the headspace (triplicates). When glucose had been totally consumed and butyric acid produced (glycolysis), the headspace was flushed with 100% CO for 5 min up to a final CO pressure of 1.8 bar. CO acts as reducing power to stimulate butanol production from endogenous butyric acid by the enriched Clostridium bacteria. CO was added at day 5, 9 and 11.Endogenous butyric acid production from glucose was first tested in 125 ml serum bottles with 30 ml medium, and with initially 5 g/L glucose and N2 or CO, i.e., Glucose + N2 and Glucose + CO. In the Glucose + N2 experiment, when glucose was totally consumed and butyric acid accumulated ((G + N)1), the headspace was flushed with CO for 5 min to reach a gas pressure of 1.8 bar at 180, 252, 360 and 432 h, respectively, to stimulate butanol accumulation ((G + N)2). In CO and glucose co-fermentation, CO was flushed at 0, 180, 276 and 432 h in the Glucose + CO experiment.The sequential conversion of glucose (glycolysis) and then CO (WLP) or the co-fermentation of glucose and CO by the enriched sludge were further investigated in two gas fed reactors . pH was 2 experiment, when glucose was totally consumed and butyric acid accumulated ((G + N)1) and at the end of the incubation ((G + N)2) after CO was added as well as two samples from the Glucose + CO experiment, when glucose was totally consumed and butyric acid accumulated ((G + C)1), and at the end of the incubation ((G + C)2).The microbial community of the four enriched sludge samples were analyzed: two samples from the Glucose + N2SO4 solution was used as mobile phase at a flow rate of 0.80 ml/min, with a sample injection volume of 20 μl, and a column temperature of 45°C  equipped with a refractive index detector, using an Agilent Hi-Plex H Column (300 × 7.7 mm). A 5 mM H of 45°C . The cel of 45°C . pH was −1 C. −1 and 74 g·moL−1.The butyric acid (HBu) conversion efficiency and butanol (BtOH) yield were calculated according to ® PowerSoil Kit  following the manufacturer’s protocol. 10 ml sludge was used for DNA extraction at the end of the incubations. The extracted DNA was quantified and its quality was checked with a Nanodrop 2000c Spectrophotometer . The extracted DNA was analyzed by Metagenomics -Seq  . The procedure has been described in DNA was extracted using a DNeasy600 of 1.5  decreased to 0.661  with a small amount of additional carbon carried over from the inoculum , half the amount of glucose was consumed after 58 h, along with the production of 70 mmoL·L product . Later, produced . CO was cubation . Both accubation .2, the relative abundance of the Clostridia and Bacilli class in the initial inoculum was, respectively, 82 and 1%, and reached, respectively, 58 and 33% when glucose was totally consumed ((G + N)1, glycolysis). Then, both decreased to, respectively, 51 and 17% and unassigned bacteria occupied 8% at the end of the incubation  2] . In the 15 to 8% .Clostridium genus level, Clostridium butyricum increased from 0.003% in the initial inoculum to 16% when glucose was totally consumed, while it decreased to 6% at the end of the incubation, after replacing the headspace with CO 1). Then, it changed to, respectively, 59 and 29% at the end of the incubation ((G + C)2) 1–9% and the Oscillospriraceae family increasing from 0.08 to 1% in (G + C)2 (In CO and glucose co-fermentation (Glucose + CO), the enriched sludge was dominated by both G + C)2) . In the (G + C)2 .Clostridium genus level, the relative abundance of C. butyricum reached 17% in (G + C)1 but it then decreased to 2% in (G + C)2 (C. carboxidivorans significantly increased to 6% in (G + C)2 compared to 0.1% in (G + C)1 2 . C. carb(G + C)1 . The rel03 to 3% .1, (G + N)2 (G + C)1 and (G + C)2. (G + N)1 had high dissimilarity with (G + C)1, while (G + N)2 had high similarity with (G + C)2, but these four enriched sludges still exhibited close distances 2 2 . Consideus level .This study showed that butanol production was enhanced in the presence of exogenous butyric acid using CO as reducing power by the enriched sludge. Butanol production was increased along with increasing exogenous butyric acid concentrations , which cC. carboxidivorans at pH 5.75 , the undissociated acid can enter the cells and may thus inhibit biomass activity and growth  than with 14 g/L HBu (3.9 g/L) ; Table 1C. ragsdalei. In that study, the reduction was independent of growth in an optimized medium with headspace CO gas exchange every 48 h and with a gas pressure of 207 kPa. C. ljungdahlii ERI-2 using syngas, CO/H2/CO2 , and reached 13.3 mM (0.99 g/L) n-butanol and 91% of the theoretical yield from butyric acid. n-butanol by a mixed culture of Alkalibaculum bacchi strain CP15 and Clostridium propionicum and obtained 0.8 g/L butanol with 1.5 g/L exogenous butyric acid, using syngas  as gaseous substrate.This study showed that the presence of acetic acid and ethanol played a positive role in accelerating butanol conversion from exogenous butyric acid. In the HAc + EtOH + HBu experiment, the butanol yield and butyric acid conversion efficiency reached 100 and 76.2%, respectively, which were both higher than in the HBu experiment . However2 during CO consumption  and CO2 was removed at the same time, alcohol production was favored  as the substrate (This study showed that endogenous butyric acid was successfully produced from glucose and CO addition triggered butanol production from the existing endogenous butyric acid  and the ubstrate . This st1 and (G + N)2 in terms of Clostridia and Bacilli classes. The Oscillibacter genus, belonging to the Clostridia class, was enriched at the end of the incubation ((G + N)2) and has been observed to be involved in acidogenesis during dark fermentation 1, while its abundance decreased compared to the inoculum entation . It was inoculum . C. carboduction . C. butyoduce H2 . The hetubstrate .−1 C, which is even slightly higher than the highest butyric acid concentration (42.9 mmoL·L−1 C) reached with only glucose (−1 C) when adding CO after endogenous butyric acid than initially adding CO (CO and glucose co-fermentation) at a pH controlled at 5.8–6.0. Co-fermentations of CO or syngas with glucose has only been reported with a few pure cultures as mentioned above, e.g., C. carboxidivorans  was consumed along with acids produced, another inorganic carbon source (CO) addition would stimulate autotrophic acetogens to convert butyric acid to butanol due to its reductant role  was higher than with Glucose + N2 (54%) 1 while Clostridium genus 46%, Oscillibacter 1% and Anaerotignum 7% in (G + N)1. The Bacilli class occupied 29% in G (G + C)2 and 17% in (G + N)2. At the Clostridium species level, the relative abundance of common autotrophic strains, such as C. carboxidivorans, C. autoethanogenum, C. ragsdalei and C. ljungdahlii, did not show much difference  . The relN2 (54%) : Clostrifference . Liu et Clostridium spp. using CO as the gaseous substrate. The presence of acetate and ethanol enhanced the butyric acid conversion efficiency compared to exogenous butyric acid. When increasing the exogenous butyric acid concentration to 14 g/L, the highest butanol concentration (3.4 g/L) was produced. Butanol accumulation was significantly enhanced up to 2.4 fold when adding CO after endogenous butyric acid production from glucose compared to when adding CO initially with glucose (co-fermentation), from which butanol was not efficiently produced with a similar amount of endogenous butyric acid production. The Clostridia and Bacilli class were dominantly enriched after glucose fermentation and at the end of the incubation. There is no big change in microbial community composition when comparing the end enriched sludge in the Glucose + CO and Glucose + N2 incubations.The exogenous supply of 3.2 g/L butyric acid resulted in the production of 1.9 g/L butanol, while in the presence of exogenous acetic acid and ethanol, butanol production was enhanced to 2.6 g/L by an enriched sludge with a high abundance of


Xenopus laevis tadpoles possess high regenerative ability and can regenerate functional tails after amputation. An early event in regeneration is the induction of undifferentiated cells that form the regenerated tail. We previously reported that interleukin-11 (il11) is upregulated immediately after tail amputation to induce undifferentiated cells of different cell lineages, indicating a key role of il11 in initiating tail regeneration. As Il11 is a secretory factor, Il11 receptor-expressing cells are thought to mediate its function. X. laevis has a gene annotated as interleukin 11 receptor subunit alpha on chromosome 1L (il11ra.L), a putative subunit of the Il11 receptor complex, but its function has not been investigated. Here, we show that nuclear localization of phosphorylated Stat3 induced by Il11 is abolished in il11ra.L knocked-out culture cells, strongly suggesting that il11ra.L encodes an Il11 receptor component. Moreover, knockdown of il11ra.L impaired tadpole tail regeneration, suggesting its indispensable role in tail regeneration. We also provide a model showing that Il11 functions via il11ra.L-expressing cells in a non-cell autonomous manner. These results highlight the importance of il11ra.L-expressing cells in tail regeneration. Xenopus laevis tadpoles have high regenerative ability, and can regenerate functional whole tails within a week of amputation5. In X. laevis tadpole tail regeneration, the spinal cord and notochord regenerate from the same tissue type in the stump, and myofibers arise from satellite cells and not from the pre-existing myofibers7. A recent single-cell resolution analysis of tail regeneration revealed no evidence for the emergence of multipotent progenitors or intermediate cell states suggesting transdifferentiation8. These findings suggest that lineage-restricted tissue stem cells or precursors are major contributors to tail regeneration in X. laevis tadpoles9. After tail amputation, these precursors are activated to generate a mass of undifferentiated proliferating cells at the wound stump to form the regeneration bud, and then differentiate to the tissues from which they are derived7. Induction of undifferentiated proliferating cells is an early and indispensable event for organ regeneration10. We previously reported a factor involved in this process; interleukin-11 (il11), which is highly expressed in the regeneration bud, but not in the intact tail or developmental tail bud11, is essential for inducing undifferentiated sensory neurons, notochord, and muscle during tail regeneration12. Immediate early expression of il11 occurs within 2 h post amputation (hpa), and undifferentiated cells of different cell lineages are induced only by il11, suggesting that it initiates regeneration12.Organ regenerative ability varies among animal species. Among vertebrates, fish and amphibians possess high regenerative ability, whereas mammals have restricted regenerative ability19. Il11 is received by the Il11 receptor complex comprising Il11 receptor subunit alpha (Il11ra) and glycoprotein 130 , a common co-receptor for the Il6 type cytokine family16. Binding of Il11 to the Il11 receptor complex activates Janus kinase (Jak), which is associated with Gp130 and phosphorylates Stat3 , and then phosphorylated Stat3 (P-Stat3) translocates to the nucleus to activate downstream transcription19.Il11 is a member of the interleukin-6 (Il6) type cytokine family, whose signaling cascade is well described in mammalsX. laevis has a gene annotated as interleukin-11 receptor subunit alpha on chromosome 1L (il11ra.L), its function as an Il11 receptor has not been investigated. We performed a functional analysis of il1ra.L in culture cells and in vivo, and an expression analysis of intact tadpoles and regenerating tails. We offer a possible model of il11ra.L-expressing cell function in tadpole tail regeneration.Il11 activity in tissue regeneration is thought to be elicited by cells receiving the secreted Il11, and thus we hypothesized that Il11 receptor-expressing cells play pivotal roles in regeneration. Although X. laevis Il11ra.L functions as an Il11 receptor component. Il11 elicits Stat3 phosphorylation and its nuclear translocation in mammals19. We therefore examined whether P-Stat3 nuclear translocation occurs when X. laevis Il11 is applied to X. laevis cells expressing il11ra.L, and whether P-Stat3 nuclear translocation is abolished by il11ra.L knockout (KO).First, we investigated whether 20, derived from X. laevis tadpole. In mammals, signal transduction to Stat3 phosphorylation in response to Il11 occurs via the Il11ra and Gp130  complex, JAK and STAT318. The corresponding genes in X. laevis are il11ra.L, il6st.L, il6st.S, jak1.L, jak1.S, jak2.L, jak2.S, stat3.S, and stat3.L, and reverse transcription-polymerase chain reaction (PCR) confirmed that these genes are expressed in XTC-YF  having different il11ra.L target sites . The mRNAs have a signal peptide sequence and a 3 × flag tag sequence  and XTC-YF -YF Fig. A. We syntes Fig. . ProteinX. laevis Il11ra.L also acts as an Il11 receptor component in tadpole tail regeneration. Knockdown (KD) of il11 impairs tail regeneration in tadpoles12. Thus, we examined whether KD of il11ra.L also impairs tadpole tail regeneration.We next investigated whether cas9 mRNA and il11ra.L guide RNA #1 (#1 KD); (2) embryos injected with cas9 mRNA, and both guide RNAs #1 and #2 to improve KD efficiency (#1&#2 KD); and (3) a control group in which the embryos were injected with only cas9 mRNA. We selected normally developed tadpoles 4 days post fertilization  and amputated their tails. A heteroduplex mobility assay25 using amputated tails confirmed that the gene editing was successful  embryos injected with ful Fig. . Tail re(#1 KD);  embryos (#1 KD);  embryos (#1 KD);  embryos (#1 KD);  embryos (#1 KD);  embryos (#1 KD);  embryos ps Table , suggestil11ra.L KD affects other early signaling events of regeneration, we compared gene expression profile changes after tail amputation between the cas9 and il11ra.L KD groups. Tail stumps of tadpoles at 0 and 24 h post amputation (hpa) in the cas9 and il11ra.L KD groups were sampled, and RNA-sequencing (RNA-seq) was performed. We screened those genes that were significantly more upregulated in the 24 hpa cas9 sample compared with the others; i.e. genes for which 1) expression did not differ significantly between the cas9 0 hpa samples and the KD 0 hpa samples, suggesting that their developmental expression was not affected by il11ra.L KD, 2) expression was higher in cas9 24 hpa samples than in cas9 0 hpa samples, suggesting that their expression was upregulated by tail amputation, and 3) expression did not differ significantly between 0 and 24 hpa of the KD samples, suggesting that the upregulation by tail amputation was abolished in the il11ra.L KD tadpoles. The screening identified 6 genes exhibiting the described expression pattern , or if it is received by some other cells that subsequently activate tissue stem cells or precursors to generate undifferentiated cells .We previously reported that il11ra.L KD tadpoles by injecting mRNA and guide RNA #1 into 1-cell stage embryos , and homozygously KO (referred to as “KO”) cells. il11ra.L KD tadpoles had a normal morphology and survival rate , il11ra.L KO progenitors are activated like WT or HT cells, and these KO cells contribute to form the regenerated tail. To test this, we generated il11ra.L KD tadpoles and amputated their tails at 4 dpf. Amputated tails were sampled as “developmental tails”. Amputated tadpoles were maintained for 7 days and regenerated tails were sampled as “regenerated tails”. We extracted genomic DNA from these tail samples, and compared the mutation (insertion/deletion) ratio at the guide RNA #1 target site of the il11ra.L genomic locus of developmental tails with that of regenerated tails in the same individual. If il11ra.L KO cells do not contribute to form regenerated tails, the mutation ratio of the regenerated tail would be lower than that of the developmental tail, which contains KO cells.We performed the following experiments Fig. . We genete Table , and theil11ra.L KO precursors contribute to form regenerated tails and that these precursors do not require cell autonomous il11ra.L expression for tail regeneration. Although the mode of Il11 function might differ according to the tissue type, we detected no significant decrease in the mutation ratios, supporting the idea that at least the tissues comprising a large portion of the regenerated tails are formed in a way depicted by the “Indirect model”.The mutation ratio of the developmental tails was not significantly different from that of the regenerated tails Fig. , suggestil11ra.L, whose function as an Il11 receptor has not been verified experimentally in X. laevis, is required for the nuclear localization of P-Stat3 in response to Il11 in cultured X. laevis cells  XTC-YF is less sensitive to Il11, which might be due to the relatively low expression of il11ra.L  wounding; il11ra.L-expressing cells throughout the body are ready to respond to Il11 secreted from wounds in any body part. It is unclear, however, whether all or only some il11ra.L-expressing cells have the potential to respond for regeneration.Il11 signaling is mediated by Il11ra.L, indicating that ils Fig. . Tail amil11ra.L is required for tail regeneration  animal is required.Although ion Fig. , we deteles Fig. . These rX. laevis, both il1112 and il11ra.L , but this capacity is lost by P7 with the development of fibrosis after apical resection37. Whereas induction of il11 expression in organ regeneration or wound healing is conserved across animal species, it is possible that the downstream events of Il11, which are elicited via il11ra expressing cells, differ between regeneration-capable or incapable species/developmental stages/tissues. Our study highlights the pivotal role of il11ra in organ regeneration and provides a basis for future studies of the role of Il11 in organ regenerative ability.In Xenopus laevis adults were purchased from domestic breeders  and maintained in fresh water at 20 °C. Animal experiments were carried out in accordance with the Guidelines for Proper Conduct of Animal Experiments of Science Council of Japan. The protocols of experiments using living modified organisms in this study were approved by the Committee on living modified organisms experiments of the Graduate School of Science at the University of Tokyo (DNA Exp. 17–3). Experiments and data analysis procedures were performed in compliance with the ARRIVE guidelines (https://arriveguidelines.org).X. laevis tadpoles20  was maintained in 0.5 × L-15 medium for cultured cells , 10% fetal bovine serum , 2 mM L-glutamine , and 1% penicillin–streptomycin mixture ) at 25 °C.The XTC-YF cell line derived from For RT-PCR, total RNA was extracted from XTC-YF using TRIzol Reagent . Reverse transcription was performed using a PrimeScript RT reagent Kit with gDNA Eraser . A group without reverse transcription (RT-) was also prepared as a control. Sequences of primers used are shown in Table For qRT-PCR, total RNA was extracted from 3 lots of XTC-YF using RNeasy Mini Kit . Reverse transcription was performed using a PrimeScript RT reagent Kit with gDNA Eraser. A group without reverse transcription (RT-) was also prepared as a control. Sequences of primers used are shown in Table cas9 system was used. The il11ra.L guide RNAs were designed with CRISPRdirect38. The target sequences were as follows: #1, GGTGAGAAGCACAATCACC[CGG]; and #2, GGGAGCAGCGTCTCAACAC[TGG] (sequences in brackets indicate the protospacer adjacent motif). The target sequences were inserted into a DR274 plasmid39 and guide RNAs were synthesized using an AmpliScribe T7-Flash Transcription Kit  before purification with an RNeasy Mini Kit. For cas9 mRNA synthesis, pXT7-hcas940  was digested with Xba I (Takara) and then mRNA was synthesized using an mMESSAGE mMACHINE T7 ULTRA Kit  and purified with an RNeasy Mini Kit (QIAGEN).The CRISPR/il11ra.L guide RNA #1 or #2 and cas9 mRNA were transfected into XTC-YF cells using Lipofectamine LTX Reagent (Thermo Fisher Scientific). After cloning by limiting dilution, cells of cloned cultures were suspended in 50 mM NaOH at 98 °C for 10 min, and neutralized with 1/10 vo1ume of 1 M Tris–HCl pH 7.5 to obtain genomic DNA extract. Regions including the guide RNA target site were amplified by PCR with primers . The obtained sequences are shown in Fig. X. laevis oocytes. The construction procedures are shown in Fig. il11.L sequence (AB933563) was amplified and the 3 × flag tag sequence was inserted between the il11.L signal peptide and mature peptide sequences  and amplified, the flag sequence was added to the C-terminus, and the mRNA was synthesized , or enzymatically by treating oocytes in 0.5 × L-15 medium for oocytes  supplemented with 25 mM HEPES [pH 7.6], 10 mM CaCl2, and 10 mg/ml collagenase  for 1 h. Oocytes were maintained overnight in 0.5 × L-15 medium for oocytes at 20 °C. We injected 50.6 nl of 1 ng/nl mRNA into oocytes. For the control group, an equal volume of RNase-free water was injected. Injected oocytes were maintained overnight and then the medium was changed. After 3 or 4 days, the oocyte culture supernatants containing the secreted protein were collected.Oocyte injection was performed essentially as described previouslyThe supernatants were subjected to glycine-SDS-PAGE and concentrations of synthesized proteins were estimated on the basis of the intensity of CBB stained bands that were detected near the expected molecular size, and bands of a series of known amounts of BSA, using ImageJ. For Western blotting, mouse anti-DYKDDDDK (FLAG) antibody  or mouse IgG2b isotype control  was used as the primary antibody, and Clean Blot™ IP Detection Reagent (HRP) (Thermo Fisher Scientific) was used as the secondary antibody. The signal was detected by chemiluminescence using ECL select , and ImageQuant LAS 4000 (Cytiva).il11ra.L knock-out cell lines were seeded on 96-well plates. After serum starvation in a serum-free medium (0.5 × L-15 medium for cultured cells without FBS) for 4 h, the oocyte culture supernatant containing recombinant proteins was added to the medium. The final concentrations were 1800 ng/ml, 300 ng/ml, and 50 ng/ml for Il11 and GST; and 1800 ng/ml for Lif. After 20 min, cells were fixed with 4% paraformaldehyde/PBS and then permeabilized with cooled methanol. Anti-phospho-STAT3 (Tyr705) antibody  and Alexa Fluor 555-conjugated anti-mouse IgG goat antibody  were used to stain phospho-STAT3. Nuclei were stained with 1 µg/ml DAPI. Cells were observed using a fluorescence microscope BZ-X800 .XTC-YF wild-type and cas9 mRNA prepared as described above were used. Injection was performed essentially as described previously44. Fertilized eggs were dejellied with 3% cysteine and divided into 3 groups; 18.4 nl of (1) cas9 mRNA (700 ng/µl), (2) cas9 mRNA (700 ng/µl) and il11ra.L guide RNA #1 (40 ng/µl), (3) cas9 mRNA (700 ng/µl), il11ra.L guide RNA #1 and #2  were injected into the animal hemisphere of the egg. Injected embryos were maintained at 12 °C for 6 h and reared at 20 °C. Tadpoles of 4 dpf (stage 41) were anesthetized with 0.02% MS-222  at 0.1 × Steinberg solution 2, 83 µM MgSO4, 300 µM HEPES, pH7.4), and the tails were amputated. Tadpoles with amputated tails were kept in 0.1 × Steinberg solution 20 °C for 7 days, and then classified into 4 groups depending on tail regeneration . Statistical significance was assessed by Fisher’s exact test with the Benjamini–Hochberg method45. For measuring the regenerated tails, we took photos of the regenerated tails of cas9 and #1&#2 KD tadpoles at 7 dpa and the parameters shown in Fig. 46.Guide RNAs and  ng/µl),  cas9 mRNA schematic of HMA is shown in Fig. il11ra.L KD (using gRNA #1 and #2) tadpoles and control tadpoles (no gRNAs) were generated as described above. The tails of 4 dpf tadpoles were amputated, and the amputated tail stumps (tail tips were removed) were collected as 0 hpa samples. Tadpoles with amputated tails were kept for 24 h and the tail stumps were collected as 24 hpa samples. We collected 3 lots of 17–24 tail stumps, and total RNA was extracted using the RNeasy Mini Kit. RNA-seq was performed at Genome-Lead Co., Ltd. . The mRNA was isolated using KAPA mRNA Capture Kit , followed by production of cDNA libraries using MGIEasy RNA Directional Library Prep Set . RNA-seq was performed using DNBSEQ-G400 RS (MGI) with the DNBSEQ G400RS High Throughput Sequencing Set, to generate approximately 4 × 107 paired-end reads (150 bp × 2) from each cDNA library.X. laevis genome version 9.1 sequence (Xla.v91.repeatMasked.fa) using HISAT2 v2.1.047, and the number of reads mapped to each gene was counted using FeatureCounts v2.0.148 with a gene model (XL_9.1_v1.8.3.2.primaryTranscripts.gff3). The genome sequence and gene model were downloaded from Xenbase. Differential expression analysis was performed using baySeq package v2.26.049.Obtained reads were mapped to il11ra.L KD tadpoles with guide RNA #1 as described above. Tails of 4 dpf tadpoles were amputated, and amputated tails were sampled as “developmental tails”. Amputated tadpoles were kept separately in 24 well plates (1 tadpole per well) for 7 days, and then regenerated tails were sampled as “regenerated tails”. Genomic DNA was extracted as described above. PCR with primers (sequences are shown in Table 50 (https://ice.synthego.com/) to estimate the mutation ratio.We generated il11ra.L coding sequence was amplified by PCR with primers . Digoxigenin-labeled RNA probes were synthesized with T7 or SP6 RNA polymerase (Roche) and DIG RNA Labeling Mix (Roche).For RNA probe synthesis, a part of the 51 with several modifications. Wild-type intact 4 dpf tadpoles, and wild-type 1, 3, 5 dpa tadpoles which were amputated at 4 dpf, were fixed in MEMFA fixative (0.1 M MOPS (pH7.4), 2 mM EGTA (pH8.0), 1 mM MgSO4, and 10% formalin) at room temperature for 48 h. They were dehydrated with an ethanol and Clear Plus  series and embedded in Paraplast plus (Merck), and 7-µm thick sagittal sections were prepared. Sections were rehydrated, permeabilized with 20 µg/ml proteinase K (Roche) and 1% Triton X-100, followed by acetylation with 0.25% acetic anhydride. The digoxigenin-labeled probe were hybridized at 58 °C for 20 h, and washed with 0.3 × SSC . The slides were incubated with alkaline phosphatase conjugated anti-digoxigenin antibody (Roche). Signals were detected with NBT/BCIP stock solution (Roche) for 24 h. To bleach the endogenous melanin pigments, slides were placed in 0.5 × SSC containing 0.3% H2O2 under fluorescent light overnight. The slides were observed using a differential interference microscope.Tadpoles were obtained by artificial fertilization and maintained in 0.2% sea salt water at 20 °C. In situ hybridization was performed as described previouslySupplementary Information.


Dynamic changes of histone posttranslational modifications are important contexts of epigenetic reprograming after fertilization in pre-implantation embryos. Recently, lactylation has been reported as a novel epigenetic modification that regulates various cellular processes, but its role during early embryogenesis has not been elucidated.2), gradient oxygen concentration (5% O2 to 2% O2) and embryos obtained from in vivo, but the modifications were greatly reduced in embryos cultured in hypoxic condition (2% O2). In contrast, nuclear accumulation of H3K18ac or H3K23ac was not significantly affected under hypoxic condition. Moreover, the developmental rate of in vitro cultured embryo was significantly reduced by low oxygen concentration and small molecule inhibition of LDHA activity led to decreased lactate production, as well as reduced histone lactylation and compromised developmental rate.We examined nuclear accumulation of H3K23la, H3K18la and pan histone lactylation in mouse oocytes and pre-implantation embryos by immunofluorescence with specific antibodies. All of the three modifications were abundant in GV stage oocytes, and both H3K23la and pan histone lactylation could be detected on the condensed chromosomes of the MII oocytes, while H3K18la were not detected. After fertilization, the nuclear staining of H3K23la, H3K18la and pan histone lactylation was faint in zygotes but homogeneously stained both of the parental pronuclei. The signal remained weak in the early cleavage stage embryos and increased remarkably in the blastocyst stage embryos. Comparison of the embryos cultured in four different conditions with varying concentrations of oxygen found that H3K23la, H3K18la and pan histone lactylation showed similar and comparable staining pattern in embryos cultured in atmospheric oxygen concentration (20% OWe provided for the first time the dynamic landscape of H3K23la, H3K18la and pan histone lactylation in oocytes and pre-implantation embryos in mice. Our data suggested that histone lactylation is subjected to oxygen concentration in the culture environment and hypoxic in vitro culture reduces histone lactylation, which in turn compromises developmental potential of pre-implantation embryos in mice.The online version contains supplementary material available at 10.1186/s13072-021-00431-6. Botrytis cinerea, and is involved in central carbon metabolism and protein biosynthesis in Oryza sativa [Gamete genomes undergo widespread epigenetic reprogramming to restore totipotency in zygotes , 2. Covaa sativa , 17. Fura sativa .The development of pre-implantation embryos depends on appropriate reproductive tract environment , of whicOxygen concentration regulates early embryonic development through fine-tuning expression of important transcription factors and genes related to glycolysis pathway . CellulaIn this study, we characterized the nuclear accumulation of H3K18la, H3K23la and pan histone lactylation in oocytes, zygotes and pre-implantation embryos in mice for the first time, and further explored the relationship between oxygen concentration and the lactylation modifications in pre-implantation embryos. Inhibition of the Lactate dehydrogenase A (LDHA) activity reduced histone lactylation level and impaired developmental rate of the pre-implantation embryos. Our findings suggested that hypoxic in vitro culture reduces histone lactylation, which in turn impairs pre-implantation development in mice.Histone lysine lactylation (Kla) as a novel epigenetic modification has been recently reported to play important roles in different cellular processes , and we It has been well documented in mammalian zygotes that the paternal and maternal genomes remain physically separated after fertilization  and epigOxygen has been shown to regulate energy metabolism and gene expression during pre-implantation embryo development . LactateTo confirm the hypoxic culture conditions, we used specific antibodies to examine the expression of the well characterized hypoxia inducible genes, HIF1α and HIF2α , in the 2) is significantly lower than that of atmospheric oxygen concentration (~ 20% O2)  Fig. F, indicaTo investigate the effect of oxygen concentrations on embryonic development, in vitro fertilized oocytes were cultured under different oxygen concentrations as shown in the Fig. n = 3) in the GSKA treated group, which was reduced by 7.1% compared to the control group   , a speci 3) Fig. A, demons 3) Fig. B. Immuno 3) Fig. C. In add 3) Fig. D, E. TheEpigenetic dynamics regulates gene expression in a spatial and temporal manner to safeguard developmental potential and cell lineage specification in the pre-implantation embryos , 38. Lac2) [Oxygen plays an important role in embryonic development by regulating epigenetic dynamics and energy metabolism , 43. Hyp2) , and we 2) , 48. How2) , indicat2) , 51. Ind2) , 53. TheWhen transferred from 7 to 2% oxygen concentration at morula stage and cultured onward, the proportion of inner cell mass cells was increased in bovine blastocyst stage embryos . SimilarAccumulating evidences showed that crosstalk between metabolism and epigenetics plays important roles in gene expression, cell proliferation and cell differentiation , and hasIn this study, we provided for the first time the nuclear staining landscape of histone lactylation in oocytes and pre-implantation embryos in mice. Pan histone lactylation, H3K23la, and H3K18la were all abundant in oocytes and pre-implantation embryos, and reach the highest level in the blastocyst stage embryos, suggesting their potential involvement during pre-implantation development. Our data also suggested that histone lactylation is correlated with cellular lactate level in the embryos and subjected to variation of oxygen concentration in the culture environment. Moreover, hypoxic in vitro culture reduces histone lactylation and compromises developmental potential of pre-implantation embryos.ICR mice used for oocytes/embryos collection were purchased from Vital River Laboratory , and were housed and bred in a specific pathogen-free facility in the Inner Mongolia University. The mice were kept at a constant temperature of 22 ± 2 °C on a 12 h light/dark cycle and had unrestricted access to chow and water.2) from zygote to blastocyst stage; (2) in vivo group: embryos were collected by flushing oviduct and uterine horns at specific timepoint as mentioned above; (3) oxygen gradient group: in vitro fertilized oocytes were cultured at 5% oxygen concentration for 3 days and then changed to 2% oxygen concentration; and (4) hypoxia group: in vitro fertilized oocytes were cultured in 2% oxygen concentrations from zygote to blastocyst stage. The inhibitor GSK2837808A (GSKA) was dissolved in DMSO and added to KSOM medium to inhibit lactate dehydrogenase A (LDHA) activity. As a control, embryos were incubated with the matching concentration of DMSO. The developmental potential of 4-cell, morula and blastocyst stage embryos was assessed morphologically under a Leica dissection microscopy at 48 hpi, 72 hpi and 96 hpi, respectively.Female ICR mice, ~ 8 weeks, were super-ovulated by intraperitoneal (i.p.) injection of 10 IU pregnant mare serum gonadotropin  followed by 10 IU human chorionic gonadotropin  ~ 48 h later. For in vivo embryo collection, super-ovulated female mouse was caged individually with one fertile male overnight. 13 h post-hCG (phCG) injection, mating was confirmed by the presence of the vaginal plug. Embryos were obtained by flushing the fallopian tubes and uterine horns with M2 medium. 2-cell, 4-cell, morula and blastocyst stage embryos were collected at 42–44, 51–53, 84–86 and 92–94 h post-hCG injection, respectively. For in vitro oocytes/embryos collection, metaphase II phase (MII) oocytes were harvested from the ampulla of the oviducts at 14–16 phCG in human tubal fluid (HTF) medium. Sperm were obtained from the caudal epididymis of adult male mice and capacitated for 1.5 h in HTF medium, and the sperm were co-incubated in HTF for 2 h for in vitro fertilization. Fertilized oocytes were cultured till blastocyst stage in KSOM  medium under different oxygen conditions. Zygotes were collected at 2, 4, 6, 8, and 10 h post-insemination (hpi). Embryos were collected at 24 hpi (2-cell), 48 hpi (4-cell), 72 hpi (morula) and 96 hpi (blastocyst), respectively. Embryos cultured or obtained from 4 different conditions were compared: (1) atmospheric oxygen group: in vitro fertilized oocytes were cultured in atmospheric oxygen concentration  containing 4% (w/v) paraformaldehyde (PFA) for 20 min at room temperature, and washed three times with 1% BSA/PBS. Then, the cells were permeabilized with 0.5% Triton X-100, 1% BSA/PBS for 20 min, and washed three times with 1% BSA/PBS. The samples were blocked with 0.1% Triton X-100, 1% BSA/PBS for 30 min and incubated with primary antibodies overnight at 4 °C. Oocytes/embryos were incubated with secondary antibodies for 1 h in dark and washed with 1% BSA/PBS for 5 min, three times. Then the samples were stained with 4,6-diamidimo-2-phenylindole (DAPI) for 10 min. Finally, the samples were mounted on slides and immunofluorescence images were taken under a Carl Zeiss LSM710 laser scanning confocal microscopy. Semi-quantitative analysis of fluorescence signals was conducted using Image J, ROI Manager Tool. The cytoplasmic pixel value of the protein was subtracted from the nucleus pixel value of the protein as the fluorescence intensity value.Primary antibodies and the dilution: anti Pan-Kla  1:500, anti-H3K23la  1:500, anti-H3K23ac  1:500, anti-H3K18la  1:200, anti-H3K18ac  1:200, anti-HIF1α  1:200, anti-HIF2α  1:200. Secondary antibodies and the dilution: anti-rabbit IgG Fab2 Alexa Fluor (R) 488 Molecular Probes  1:600; DyLight 594-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson IR) 1:100.2O was incubated in LightCycler480 real-time PCR system  and amplified using two-step conditions: 95 °C for 30s; 40 cycles of 95 °C for 5s and 60 °C for 20s. All amplifications were done in technical duplicate and biological triplicate and data were analyzed using LightCycler 96 SW 1.1 software. 18s rRNA was used as the internal control and relative expression levels of the target genes were calculated by 2−ΔΔCT method. The primer sequences for the LDHA, standard 18s rRNA are listed as below: LDHA sense: 5′-TGT CTC CAG CAA AGA CTA CTG T-3ʹ; LDHA anti-sense: 5′-GAC TGT ACT TGA CAA TGT TGG GA-3ʹ; 18s rRNA sense: 5′-CTG CCC TAT CAA CTT TCG ATG GTA G-3ʹ; 18s rRNA anti-sense: 5′-CCG TTT CTC AGG CTC CCT CTC-3ʹ.RT-qPCR was done as previously reported . Briefly2O, and solvent B, 100% MeOH. The gradient elution was programmed as follows: 0 min, 10% B; 3 min, 100% B; 4 min, 100% B and Post time, 2.0 min. The flow rate was kept at 0.3 ml/min and the injection volume was 3 μL. For quantification of the lactate, multiple reactions monitoring (MRM) mode was used with negative polarity. The MRM conditions were optimized using L-lactate standards. The MW (90), Precursor ion (89), Product ion (43.2), Fragmentor (50), and Collision energy (10) were kept constant throughout the analysis. For ESI, the ion source, parameters were set as below: capillary, 4 kV; gas temperature, 350 °C; gas flow, 11 L/min; nebulizer, 45 psi.After washing with PBS, the cells were repeatedly frozen and thawed in liquid nitrogen and 37 °C water bath to extract intracellular lactate. The samples were analyzed by UPLC-QQQMS, and separated by an Agilent ZORBAX SB-C18 RRHT column  at 30 °C. The mobile phase was composed of solvent A, 0.1% formic acid in Ht test was used for two-group comparisons. For comparison between multiple groups, two-way analysis of variance (ANOVA) was performed followed by Tukey's multiple comparisons test [p values less than 0.05 was used to determine significant difference. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.All experiments were repeated at least three times and results were expressed as mean ± SEM (standard error of the mean). More than 30 blastomeres were analyzed in each replicate of fluorescence intensity quantification and more than 20 embryos were analyzed in each replicate for the assessments of embryonic development and relative gene expression. Unpaired Student’s ons test . The datAdditional file 1. Figure S1.Additional file 2. Figure S2.Additional file 3. Figure S3.Additional file 4. Figure S4.