To improve breadth, we linked the light chain of CAP256V2LS, an optimized version of CAP256-VRC26.25 currently under clinical evaluation, to the llama nanobody J3, which has broad CD4-binding site-directed neutralization. The J3-linked bispecific antibody exhibited improved breadth and potency over both J3 and CAP256V2LS, indicative of synergistic neutralization. The cryo-EM structure of the bispecific antibody in complex with a prefusion-closed Env trimer revealed simultaneous binding of J3 and CAP256V2LS. We further optimized the pharmacokinetics of the bispecific antibody by reducing the net positive charge of J3. The optimized bispecific antibody, which we named CAP256.J3LS, had a half-life similar to CAP256V2LS in human FcRn knock-in mice and exhibited suitable auto-reactivity, manufacturability, and biophysical risk. CAP256.J3LS neutralized over 97% of a multiclade 208-strain panel (geometric mean concentration for 80% inhibition (IC This result has set off a search for other broadly neutralizing antibodies, capable of neutralizing at this potency.Antibody-mediated prevention (AMP) of HIV-1 infection has been a long-sought goal. AMP clinical studies of VRC01 demonstrate the ability of passively delivered antibodies to prevent HIV-1 infection,2 on a 208-strain panel, V2-apex-directed antibodies such as PG9 and PG163 neutralize at a mean IC80 of 0.34 and 0.113 μg/ml, respectively, PGT1454 and its somatic variant PGDM14005 neutralizes at 0.343 and 0.049 μg/ml, respectively, and CAP256-VRC26.256 neutralizes at 0.035 μg/ml. However, the neutralization breadths of these antibodies have been less than ideal, ranging from 50% to 80% on our 208-strain panel. Multispecific antibodies could improve both breadth and potency, if the right combination could be identified  to enable simultaneous engagement of multiple sites of vulnerability, including the potent V2 apex site and other sites more conserved among multiple clade viral strains.Antibodies against the second hypervariable region (V2), at the trimer apex, are among the most potent broadly neutralizing antibodies identified to date;8 or V1V2 scaffolds9 reveal recognition to occur via an extended 3rd heavy chain complementarity-determining region (CDR H3), leaving the light chain relatively unencumbered, a potentially ready site for conjugation. Indeed, among the over 2000 non-redundant structures of antibodies bound to antigen, V2-apex-directed antibodies have the longest distance between antibody-framework and antigen, with CAP256-VRC26.25 being the furthest from the antigen.10 One strategy to design bispecific antibodies is to link the light chain of these V2-apex-directed antibodies to a broadly neutralizing antibody targeting conserved sites other than the V2 site of vulnerability. The membrane-proximal external region (MPER) is well conserved and MPER-directed antibodies, such as 10E8, are very broad and potent.11 CD4-binding site-directed antibodies are suitable for fusion to V2-apex-directed antibodies as bispecific antibodies because the CD4-binding site is close to V2-apex and is more conserved. Several broadly neutralizing antibodies and nanobodies targeting CD4-binding sites, such as VRC0113 and J3,15 are available.Structures of PG9, PGT145, and CAP256-VRC26.25 in complex with HIV-1 envelope (Env) trimer80 of less than 50 μg/ml, and 77% of the 208-strain panel and 82% of the clade C panel at IC80 of less than 1 μg/ml.Here, we generated bispecific antibodies by genetically fusing single-chain variable regions (scFvs) of human antibodies or single variable domains of heavy-chain-only antibodies (nanobodies) to the light chain N terminus of CAP256V2LS, an optimized version of CAP256-VRC26.25. We assessed binding and neutralization for these bispecific antibodies and structurally characterized the broadest and most potent. We improved pharmacokinetics of these bispecific antibodies by reducing their surface electropositivity and assessed their physical properties, neutralization, manufacturability, and biophysical risk. Overall, we identify CAP256.J3LS, a bispecific antibody with a suitable half-life, capable of neutralizing 97% of a 208-virus multiclade panel and 100% of a clade C panel at an IC6 in complex with a prefusion-closed envelope trimer7 reveals that the light chain of this antibody does not interact directly with Env, leaving its N terminus free for conjugation (16–18 with nanobody J3 or scFv antibodies 10E8 and VRC01 that target non-V1V2 sites (The published structure of the super-potent antibody CAP256-VRC26.25jugation . We desi19 Tested strains included CAP256 (from clade C), JRFL (B), 426C-WITO (B), as well as a K169V variant of CAP256 with reduced binding of CAP256-VRC26.25; we also tested the ability of the light-chain variants to neutralize CAP256 and WITO.33, with simian immunodeficiency virus (SIV) as a negative control  in complex with BG505 DS-SOSIP.644 trimer visible . The lin visible , indicat visible . Binding visible ; however visible .Figure Overall, the structure confirmed the ability of the CAP256V2LS-J3-3 to bind simultaneously to both V2 apex and CD4-binding site, with two other copies of the bispecific antibody binding through only their J3 moieties to the other two CD4-binding sites of the trimer.18 We observed the bispecific antibody to have reduced half-life relative to CAP256V2LS in neonatal Fc receptor (FcRn)-knock-in mice . We analyzed the surface accessibility of Lys and Arg residues on J3 and mutated the surface-exposed residues one at a time or in combinations  and could be purified following the same steps as its parent antibody CAP256V2LS, as the additional J3 component was only attached to the light chain. Isothermal titration calorimetry (ITC) with BG505 DS-SOSIP.664 showed improved affinity for CAP256.J3LS relative to CAP256V2LS and J3 .80 of 0.08 μg/ml , VRC07-523LS (0.42 μg/ml), and N6LS (0.25 μg/ml). Finally, we carried out a manufacturability and biophysical risk assessment, and found CAP256.J3LS to be mostly low risk, except for slight colloidal instability observed at high concentration or with dynamic light scattering (DLS) .We assessed the neutralization on a 208-strain panel  and a 10in vitro affinity maturation.25 CAP256.J3LS also compared favorably with other engineered bispecific antibodies, as well as with other V2 neutralizers.27 One of the broadest and most potent V2 neutralizers, PGDM1400,27 neutralizes 78% of the 208-strain panel with IC80 < 50 μg/ml at a geometric mean IC80 of 0.069 μg/ml, whereas CAP256.J3LS neutralized with a breadth of 97% with a geometric mean IC80 of 0.035 μg/ml. The bispecific antibody BISC-1C,26 which combines CAP256-VRC26.25 with PGT128, neutralizes all 15 strains tested with an IC80 of 0.032 μg/ml, whereas CAP256.J3LS neutralized these 15 strains with an IC80 of 0.026 μg/ml and neutralized 10 of the 15 strains more potently.In this study, we created a bispecific antibody by linking the light chain of CAP256-VRC26.25 with the J3 nanobody and optimizing half-life to create CAP256.J3LS, a variant with improved breadth, potency, and half-life. CAP256.J3LS compared favorably with other CAP256 variants, such as those obtained by Farzan and colleagues through 28 It will be interesting to test the impact of CAP256.J3LS clinically.Bispecific antibodies can face manufacturing issues arising from their having two different heavy chain-light chain pairings. A nanobody-antibody bispecific, made by appending the light chain to the C terminus of the nanobody, would bypass these issues because the bispecific antibody would remain symmetric, with both arms having light chain-linked nanobodies. Here, we show that such a bispecific nanobody-antibody can be produced with high yield and, further, can show substantial neutralization synergy, neutralizing better than both antibody and nanobody parental components for over half the 208 viruses tested. Further multimerization, combining CAP256.J3LS with other antibodies, is certainly possible, although, because of the orientation specificities required for synergistic binding, such a multispecific antibody would be unlikely to have other components that bind synergistically with CAP256.J3LS. Nonetheless, such a multispecific antibody would have the advantage of having multiple antibody neutralization specificities in a single product.2 for 5 days. Antibodies in clarified supernatants were purified over 0.5 mL Protein A  resin in columns. Antibodies were eluted from Protein A columns with a low pH immunoglobulin G (IgG) elution buffer (Pierce) and immediately neutralized with one-tenth volume of 1 M Tris-HCL pH 8.0. The antibodies were buffer exchanged in phosphate-buffered saline (PBS) by dialysis and then the concentration was adjusted to 0.5 mg/ml and filtered (0.22 μm) for neutralization assays. For size-exclusion chromatography analysis, 0.5 mg of 1 mg/ml antibody was injected onto the Superose 6 Increase column (Cytiva Life Sciences) on a BioRad  NGC Chromatography System Quest 10. The flow rate was set to 0.5 mL/min, and the mobile phase B was 1 × PBS (Thermo Fisher).Light chain expression constructs of CAP256V2LS and PGDM1400 antibody variants were synthesized  and cloned into pVRC8400 expression vector. For the antibody production, 0.15 mL of Turbo293 transfection reagent (Speed BioSystems) were mixed into 2.5 mL Opti-MEM medium (Life Technology) and incubated for 5 min at room temperature (RT). 50 μg of plasmid DNAs (25 μg heavy chain and 25 μg of light chain) were mixed into 2.5 mL of Opti-MEM medium in another tube. The diluted transfection reagent was added into Opti-MEM medium containing plasmid DNAs. Transfection reagents and DNA mixtures were incubated for 15 min at RT and added to 40 mL of Expi293 cells (Life Technology) at 2.5 million cells/ml. The transfected cells were cultured in a shaker incubator at 120 rpm, 37°C, 9% CO30 Briefly, 600 μg of the plasmid encoding the trimer and 150 μg the plasmid encoding human furin were mixed and used to transfect 1 L of 293 F cells using Turbo293 transfection reagent (Speed BioSystems). Cells were incubated in shakers at 120 rpm, 37°C, and 9% CO2. On the next day, 80 ml HyClone SFM4HEK293 medium and 20 ml FreeStyle™ 293 Expression Medium were added to each liter of cells. The native-like Env trimer protein was purified from the supernatant harvested on day 7 by 2G12 affinity chromatography, followed by gel filtration on a Sephadex200 16/60HL column in PBS.A non-tagged HIV Env trimer from the isolate CAP256.wk34.c80 (GenBank: KT698226.1) was stabilized and produced in transiently transfected 293 F cells as previously described.5 cells/ml in an optimized expression medium  and incubated at 37°C, 5% CO2 for 24 hours. Prior to transfection, 40 μl/well of spent medium was removed. For transient transfection, 0.15 ug of heavy-chain plasmid DNA was mixed with 0.15 ug of light-chain plasmid DNA in Opti-MEM medium (Invitrogen), and final volume 10 μl per well in a round bottom 96-well plate, followed by mixing with 10 μl/well of 0.9 μl TrueFect-Max transfection reagent (United BioSystems) in Opti-MEM medium. After incubation for 15 min at RT, 20 μl/well of DNA-TrueFect-Max complex was mixed with growing cells in the 96-well tissue culture plate and incubated at 37°C, 5% CO2. At 20 hours post transfection, each well of culture was fed with 30 μl/well of enriched expression medium . After 5 days of transfection, the antibodies in supernatants in the 96-well tissue culture plate were characterized by 96-well-formatted ELISA. Briefly, 96-well ELISA plates  were coated overnight at 4°C with 100 μl/well of 5 μg/ml lectin  in 1× PBS. Between each subsequent step, plates were washed five times with PBS-T (PBS plus 0.05% Tween 20). After being coated, various HIV-1 trimer proteins were captured onto lectin-coated 96-well ELISA plates, respectively, by an incubation of 100 μl/well of 5 μg/ml each trimer protein for 2 hours at RT followed by blocking with 200 μl/well of CelBooster Cell Growth Enhancer Medium for Adherent Cell for 1 hour at RT. After plate wash, 30 μl/well of the expressed antibody supernatant mixed with 70 μl/well of PBS was incubated for 1 hour at RT, then followed by incubation for 30 min at RT with 100 μl/well of horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody , diluted at 1:10,000 in CelBooster Cell Growth Enhancer Medium for Adherent Cell plus 0.02% Tween 20. Finally, the reaction signal was developed with 100 μl/well of tetramethylbenzidine substrate  for 10 min at RT before the addition of 100 μl/well of 0.5 N sulfuric acid  to stop the reaction. Plates were read at 450 nm wavelength , and the optical densities (OD) were analyzed following subtraction of the nonspecific horseradish peroxidase background activity. All samples were measured in duplicate.Twenty-four hours prior to the DNA-transient transfection, 100 μl/well of log-phase growing HEK 293 T cells were seeded into a flat bottom 96-well tissue culture plate (Corning) at a density of 3 × 1031 all of which yielded highly similar results.As described below, neutralization was assessed in one of four formats of the Env-pseudotyped assay,2 The following day, all wells received 100 μl of fresh cDMEM and were incubated overnight at 37°C. The following day, 50 μl of Steadylite Plus Reporter Gene Assay System (PerkinElmer) was added to all wells, and plates were shaken at 600RPM for 15 minutes. Luminometry was then performed on a SpectraMax L (Molecular Devices) luminometer. Percent neutralization is determined by calculating the difference in average Relative Light Units (RLU) between virus only wells (cells + virus column) and test wells (cells + plasma/Ab sample + virus), dividing this result by the average RLU of virus only wells (cell + virus column) and multiplying by 100. Background is subtracted from all test wells using the average RLU from the uninfected control wells (cells only column) before calculating the percent neutralization. Neutralizing plasma antibody titers are expressed as the antibody concentration required to achieve 50% neutralization and calculated using a dose–response curve fit with a 5-parameter nonlinear function.(1) Standard neutralization assays were performed in 96-well formats as follows: 10 μl of five-fold serially diluted mAbs in cDMEM was incubated with 40ul of diluted HIV-1 Env-pseudotyped virus and incubated for 30 minutes at 37°C in a 96-well CulturPlate (Perkin Elmer). 20 μl of TZM-bl cells  with or without 70 μg/ml DEAE-Dextran was then added and incubated overnight at 37°C. Each experiment plate also had a column of cells only (no antibody or virus) and a column of virus only (no antibody) as controls for background TZM-bl luciferase activity and maximal viral entry, respectively. Serial dilutions were performed with a change of tips at each dilution step to prevent carryover.(2) High-throughput screening was performed by single-point assay: supernatants from cells transfected in 96-well plates were used undiluted in the assay described above, in duplicate. Wells with >50% reduction in signal compared to baseline were scored as positive for neutralization.32 Assays were performed by microneutralization in an optimized and qualified automated 384-well format as described,31 with the modification of changing tips after each antibody dilution. Data were analyzed as above.(3) Selected monoclonal antibodies (mAbs) were assessed on a panel of 208 geographically and genetically diverse Env pseudoviruses representing the major subtypes and circulating recombinant forms.33 Neutralization assays were conducted using TZM.bl cells as previously described.31 Briefly, mAb samples were tested in duplicate in 96-well plates using a primary concentration of 50 µg/ml or 1 µg/ml and serially diluted fivefold seven times. The 1 µg/ml start value was used instead of changing tips, as noted above. The HIV-1 Env pseudovirus was added to antibody serial dilutions and plates were incubated for 1 h at 37°C. TZM.bl cells were then added at 1x104/well with DEAE-Dextran at a final concentration of 11 µg/ml. After 48-h incubation at 37°C, plates were harvested using Bright-Glo luciferase (Promega) and luminescence detected using a GloMax Navigator luminometer . Antibody concentrations that inhibited 50% or 80% of viral infection were determined . Neutralization assays were conducted in a laboratory meeting Good Clinical Laboratory Practice quality assurance criteria.(4) Select mAbs were assessed on a panel of 100 clade C Env pseudoviruses from acute infection.Each antibody sample was diluted in 1500 µL of mobile phase A (MPA), 10 mM sodium phosphate, pH 7.2 ± 0.2 to a final concentration of approximately 20 µg/mL. It was then injected onto the HiTrap 1 mL Heparin HP column (Cytiva Life Sciences) on a BioRad  NGC Chromatography System Quest 10. The flow rate was set to 1.0 mL/min, and the mobile phase B (MPB) was 10 mM sodium phosphate, 1 M NaCl, pH 7.2 ± 0.2. The column was equilibrated in 100% MPA before each injection; the gradient was as follows (1): 0–2 min, 100% MPA; (2): 2–12 min, 100% MPA to 100% MPB; (3) 12–14 min, 100% MPB. UV absorbance was detected at 280 nm using Chromlab.Autoreactivity of antibodies was evaluated using the ANA Hep-2 Test System  and anticardiolipin ELISA (Inova Diagnostics Cat. No.: 708625). Briefly, all antibodies were tested at 25 and 50 μg/ml as per the protocol from the manufacturer of the ANA Hep-2 Test System. Antibodies VRC01LS, 4E10, VRC07-523-LS, and VRC07-G54W were used as controls and scored as 0, 1, 2, and 3, respectively. HEp-2 cells were obtained from ZEUS Scientific. Slides were imaged on a Nikon Eclipse Ts2R microscope with a 20 × objective lens for 500 ms. The fluorescent signals of the test antibodies were estimated visually in comparison to the control ones. Scores over 1 at 25 μg/ml were defined as autoreactive, and between 0 and 1 as mildly autoreactive. In the cardiolipin ELISA, all the antibodies were tested at 100 μg/ml, followed by a 3-fold serial dilution. IgG phospholipid (GPL) units were derived from the standard curve. GPL score below 20 was considered as not reactive, and between 20 and 80 as low positive and greater than 80 as high positive. The reported results are representative of at least two independent experiments.Binding experiments by ITC were performed at 37°C using a MicroCal VP-ITC microcalorimeter from Malvern Panalytical . All reagents were dissolved in and exhaustively dialyzed against PBS, pH 7.4. The syringe was filled with either J3 nanobody at a concentration of ~0.3 mg/mL (~24 µM) or either CAP256V2LS IgG or bispecific CAP256.J3LS IgG at ~1.0 mg/mL (~6 µM IgG). The calorimetric cell was filled with BG505 DS-SOSIP.664 Env trimer at a concentration of ~0.2 mg/mL (~1 µM trimer) and either an IgG or a nanobody was added stepwise in 6 or 10 µL aliquots with 300 s interval during a continuous stirring at 300 rpm. The heat evolved upon each injection was obtained from the integral of the calorimetric signal, and the heat associated with binding was obtained after subtraction of the heat of dilution. The enthalpy change, ΔH, the association constant, Ka  and the stoichiometry, N, were obtained by nonlinear regression of the data to a single-site binding model using Origin with a fitting function made inhouse. The Gibbs energy, ΔG, was calculated from the binding affinity using ΔG = -RTlnKa ) and T is the absolute temperature in kelvin. The entropy contribution to the Gibbs energy, -TΔS, was calculated from the relation ΔG = ΔH -TΔS. The results were normalized per mole of nanobody or half-IgG and the stoichiometry, N, denotes the number of half-IgG per trimer.34 All mice were bred and maintained under pathogen-free conditions at the American Association for the Accreditation of Laboratory Animal Care-accredited Animal Facility at the National Institute of Allergy and Infectious Diseases and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals. All the mice were between 6 and 13 weeks of age. The study protocol was evaluated and approved by the National Institutes of Health’s Animal Care and Use Committee (ASP VRC-18-747).Human FcRn transgenic mice (FcRn-/- hFcRn (32) Tg mice, JAX stock #014565, The Jackson Laboratory) were used to assess the pharmacokinetics of wild type and Fv glycan-removed antibodies. Each animal was infused intravenously with 5 mg mAb/kg of body weight. Whole blood samples were collected at day 1, 2, 5, 7, 9, 14, and 21. Serum was separated by centrifugation. Serum mAb levels were measured by ELISA using either anti-idiotypic antibodies  as described previously.35 A set of 208 HIV-1 strains was used in the neutralization fingerprinting analysis.The neutralization fingerprint of an mAb or polyclonal plasma is defined as the potency pattern with which the antibody/plasma neutralizes a set of diverse viral strains. The neutralization fingerprints of 46 mAbs, including all described CAP256.VRC26.25 variants, were compared and clustered according to fingerprint similarity, as described previously.21 was incubated with a molar excess of CAP256V2LS-J3-3 bispecific antibody Fab fragments. A volume of 2.3 µl of the complex at 2 mg/ml concentration was deposited on a C-flat 1.2/1.3 grid (protochip.com) and vitrified with an FEI Vitrobot Mark IV with a wait time of 30 seconds, blot time of 3 seconds, and blot force of 1. Data collection was performed on a Titan Krios electron microscope with Leginon36 using a Gatan K3 direct detection device. Exposures were collected in movie mode for 2 seconds with the total dose of 51.19 e–/Å2 fractionated over 50 raw frames. cryoSPARC37 v3.3 was used for frame alignment, CTF estimation, 2D classifications, ab initio 3D reconstruction, homogeneous refinement, and nonuniform 3D refinement. Initial 3D reconstruction and final refinements were performed using C1 symmetry. The final resolution of the C1 non-uniform refinement was 3.18 Å.The BG505 DS-SOSIP.664 Env trimer7 and 7LPN15 were used for initial fits to the reconstructed map. This was followed by simulated annealing and real space refinement in Phenix v1.2038 with the sharpened map from cryoSPARC v3.3 and with a density modified map from Phenix Resolve39 and manually fit with Coot40 v0.9.8 and then improved through iterative rounds. Geometry and map fitting parameters were evaluated using Molprobity41 v4.5.1 and EMRinger.42 PyMOL v2.5 (www.pymol.org) and ChimeraX43 v1.3 were used to generate figures.Coordinates from the PDB IDs 6VTTManufacturability for CAP256.J3LS was assessed by visual inspection, dynamic light scattering, thermal transitions by dynamic light scattering, differential scanning calorimetry, circular dichroism, and isothermal chemical denaturation as previously described.Click here for additional data file.


Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5, which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt small interfering RNAs (siRNAs), heterochromatic siRNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs), respectively. The former small RNAs are linked to silencing of transposable elements and heterochromatic repeats, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient ‘eudicot-type’ DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 5′ phosphates and 3′ overhangs, consistent with the structural properties of their  Arabidopsis thaliana encodes four DCL proteins, AtDCL1–4 with precise activities. AtDCL1 produces 20 to 22-nucleotide (nt) miRNAs from miRNA precursors with more variable structures compared to animal ones, while AtDCL4 and 2 generate 21 and 22-nt siRNAs from long dsRNA substrates, respectively  and microRNAs (miRNAs) are critical players in RNA silencing pathways which regulate various biological processes including organismal development and antiviral immunity . These sectively . These sectively . In contectively . This RNectively . In shorAtDCL3 targets dsRNAs that are generated by the sequential action of two polymerases, DNA-dependent RNA polymerase IV (Pol IV) and RNA-dependent RNA polymerase 2 (RDR2) . Pol IV dcl mutants have 5′ monophosphate instead of 5′ triphosphate .,29.CitruDrosophila melanogaster Dicer-2 (Dcr-2) demonstrated that the PAZ domain has two pockets that bind the 5′ and 3′ ends of the substrate dsRNA respectively. These binding pockets are critical for the precise production of small RNAs  domain, two RNase III domains and double-stranded RNA-binding domain from N to C terminal ,36,37. Pall RNAs . In addie 3′ end . Recentlucidated ,45. The Oryza sativa DCL3 (OsDCL3) and DCL5 (OsDCL5). Our analysis elucidates how DCL3 and DCL5 have become functionally specialized after gene duplication. OsDCL3 and OsDCL5 have distinct substrate specificities for both 3′ structures and 5′ phosphate, reflecting the different in vivo functions of these proteins. Moreover, we find that the PAZ domain is a key determinant of DCL3 and DCL5 substrate specificity. These preferences explain how DCL3 and DCL5 cleave substrates from the fixed end to ensure the production of functional siRNAs. Taken together, our study provides insights into the functional differentiation of DCLs via the evolution of the PAZ domains. This provides a molecular understanding of how plants have diversified and optimized RNA silencing mechanisms through DCL gene duplication.In this study, we succeeded in preparing fully functional recombinant eudicot AtDCL3, monocot The primers used in this study are listed in Arabidopsis thaliana (Col-0) cDNA using Oligo No. 1 and 2, and cloned into TOPO® vector using pENTR™/D-TOPO™ Cloning Kit (K240020). DCL3 fragment was then introduced into pASW vector using Gateway™ LR Clonase™ II Enzyme mix (Invitrogen™ 11791021). To create the longest AtDCL3 isoform, AT3G43920.2, a 30 bp DNA fragment was inserted into the plasmid by PCR using Oligo No. 3 and 4.AtDCL3 ORF (AT3G43920.3) was amplified from OsDCL3a (OsDCL3) gene was cloned from the plasmid provided by National Agricultural and Food Research Organization (Clone ID J013008L07), using Oligo No. 5 and 6 and then assembled into pASW vector by NEBuilder Hifi DNA Assembly kit (NEB).Oryza sativa anther cDNA using Oligos No. 7–14. A DNA fragment corresponding to 1–198 nt of OsDCL5 ORF could not be amplified from anther cDNA, and was therefore synthesized by PCR using Oligos No. 15–19 according to the reference sequence of OsDCL3b (OsDCL5) (CDS) from rap-db (The Rice Annotation Project Database) . These five DNA fragments  were assembled into the pASW vector using NEBuilder Hifi DNA Assembly kit (NEB).Four DNA fragments of the OsDCL5 ORF  were amplified from A DNA fragment corresponding to the PAZ domain (2560–3030 nt) of OsDCL5 was amplified by PCR using Oligos No. 20 and 21 from pASW-OsDCL5. Another DNA fragment was amplified by PCR using Oligos No. 22 and 23 from pASW-OsDCL3. These two fragments were mixed and assembled by the NEBuilder Hifi DNA Assembly kit (NEB).A DNA fragment corresponding to the PAZ domain (2560–3030 nt) of OsDCL3 was amplified by PCR using Oligos No. 24 and 25 from pASW-OsDCL3. Another DNA fragment was amplified by PCR using Oligos No. 26 and 27 from pASW-OsDCL5. These two fragments were mixed and assembled by the NEBuilder Hifi DNA Assembly kit (NEB).A DNA fragment corresponding to the PAZ domain (2560–3030 nt) of OsDCL3 was amplified by PCR using Oligos No. 28 and 29 from pASW-OsDCL3. Another DNA fragment was amplified by PCR using Oligos No. 32 and 33 from pASW-AtDCL3. These two fragments were mixed and assembled by the NEBuilder Hifi DNA Assembly kit (NEB).A DNA fragment corresponding to the PAZ domain (2560–3030 nt) of OsDCL5 was amplified by PCR using Oligos No. 30 and 31 from pASW-OsDCL5. Another DNA fragment was amplified by PCR using Oligos No. 32 and 33 from pASW-AtDCL3. These two fragments were mixed and assembled by the NEBuilder Hifi DNA Assembly kit (NEB).Drosophila Schneider 2 cells (S2 cells) were cultured in Schneider's Drosophila Medium (Gibco) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) (Sigma) and antibiotics at 28°C, sealed with parafilm.7 cells/10 cm dish) were transfected with 10 μg pASW plasmids carrying plant DCL3 family genes  or PAZ domain chimeric genes  with 20 μl X-tremeGENE™ HP DNA Transfection Reagent (Roche) following manufacturer's instructions. The transfected cells were harvested after 72 hours for lysate preparation.S2 cells (1–1.5 × 10g for 3 min at room temperature. The cell pellet was washed by cold PBS (pH 7.4) and was centrifuged at 1500 × g at 4°C. The pellets were then weighed and resuspended in equal volumes of Hypotonic buffer  containing 5 mM dithiothreitol (DTT) and 1× EDTA-free Complete Protease Inhibitor tablets (Roche) by tapping and inverting the tubes. The suspension was incubated on ice for 15 min, and then mixed thoroughly with a vortex mixer. A cell disruption vessel (Parr Instrument Company) was used to break open the cells. The lysate was clarified by centrifugation at 17 000 × g for 20 min at 4°C. The supernatant was flash frozen in liquid nitrogen and immediately stored at −80°C in single-use aliquots.S2 cells were harvested by centrifugation using a swinging-bucket rotor at 1500 × Streptavidin Sepharose High Performance beads , equivalent to 25% of the lysate by volume, were washed with 1 ml lysis buffer , and then mixed gently with the lysate. The suspension was incubated for 1 hour at 4°C on a rotator and then washed three times with wash buffer (1× lysis buffer containing 800 mM NaCl and 1% (v/v) Triton X-100). The SBP-tagged protein was then eluted with biotin elution buffer  at 4°C on a rotator for 20 minutes, and the elution step repeated 3 times. The eluates were flash frozen in liquid nitrogen and immediately stored at −80°C in single-use aliquots after adding BSA to a final concentration of 0.2 mg/ml.32P]ATP. Strands with a 5′ monophosphate were radiolabeled with T4 polynucleotide kinase (Takara) and [γ-32P]ATP. The sense and antisense strands were heat-annealed in lysis buffer as previously described , 1 mM ATP, 0.03 U/μl creatin kinase ], and 0.1 U/μl RNasin (Promega) at 25°C. To draw time course curves for each reaction, 2 μl of the reaction mixture was taken at 5,10, 20, 30, 45 and 60 min after the reaction started. These samples were mixed with 8 μl of low-salt PK solution , and then incubated at 50°C for 10 min. An equal volume of 2× formamide dye  was then added and incubated at 95°C for 2 min. The cleavage products were analyzed on 15% denaturing polyacrylamide gels and detected by autoradiography. Small RNA products were quantitated from relative band intensities measured with a Typhoon FLA 7000 image analyzer  and quantified using MultiGauge software (Fujifilm Life Sciences). The cleavage efficiency in Figures Three nanomolar S–KOH pH .4, 12.5%in vitro, we successfully prepared full-length recombinant DCL proteins: OsDCL5, OsDCL3 and AtDCL3 using Drosophila S2 cells  were expected , 1-nt overhang (1ovr), and 2-nt overhang (2ovr)  Figure . Both At) Figure , C. At e) Figure , C. In c) Figure , showing) Figure , C. We s) Figure , C. In c) Figure . We next) Figure . These r) Figure , suggestA recent structural study showed that the 5′ nucleotide identity and the thermodynamic stability at the terminal base pairs affect the recognition of dsRNA substrates . To examin vitro dicing assays using crude plant lysates confirmed that a 5′ phosphate is required for AtDCL3-mediated cleavage of dsRNAs carrying 3′ overhangs (in vitro dicing assays with 3′ 1-nt overhang substrates radiolabeled at the 5′ monophosphate of antisense strands. These substrates carry either a 5′ monophosphate group (MonoP) or a hydroxyl group (OH) on the sense strand  or triphosphorylated (TriP) at the 5′ end of their sense strands . Gi. GiDrosoe domain , these sArabidopsis often possess A and U at the second nucleotide  as substrates for transcription and lacks binding regions for the capping complexes, Pol IV-synthesized transcripts are expected to possess a triphosphate group at the 5′ end ,51. RDR2in vitro ,16. Thus 5′ ends ,19,22, r 5′ ends . Given t 5′ ends , dephospthaliana .It is now believed that the appearance of DCL5 in monocots is explained by the ‘sub-functionalization’ of the ancestral DCL3 gene, which is speculated to function in the production of both hc-siRNAs and phasiRNAs . HoweverAll data are included in the manuscript or in the Supplementary Data and are available from the corresponding author upon request.gkac223_Supplemental_FilesClick here for additional data file.


In all cases, I2 linkers are connected with bromide ligands via halogen···halogen non-covalent interactions. Articles 1–3 were studied using Raman spectroscopy, thermogravimetric analysis, and diffuse reflectance spectroscopy. The latter indicates that 1–3 are narrow band gap semiconductors.Three supramolecular bromostannates(IV) with “trapped” diiodine molecules, Cat Metal halide complexes, or halometalates, represent the class of coordination compounds which, apart from fundamental interest , attracts attention due to numerous areas of materials science where they can be applied. Those include design of ferroelectric and ferroelastic materials ,2,3,4,5,6]2− anions. A possible strategy to overcome this problem is the use of supramolecular approaches . One of such ideas presented recently is the use of supramolecular polyhalogen-halometalate hybrids. Incorporation of di- or polyiodide units into a structure commonly yields a narrower optical band gap of resulting compounds 2− octahedral unit with ≈8% occupancy has been found  spectrometer with the excitation by the 633 nm line of the He-Ne laser. The spectra at room temperatures were obtained in the backscattering geometry with a Raman microscope. The laser beam was focused to a diameter of 2 μm using a LMPlan FL 50×/0.50 Olympus objective. The spectral resolution was 0.7 cmDiffuse reflectance spectra were measured on a setup which consisted of a Kolibri-2 spectrometer , fiber optic cable QR-400-7 , and deuterium–tungsten lamp AvaLight-DHS . The reference of 100% reflectance was BaSO4 powder. The spectra were recorded five times in the wavelength interval of 300–1000 nm and then averaged to reduce the random error.−1, the gas flow rate of 60 mL min−1, and open Al crucibles.TGA were carried out on a TG 209 F1 Iris thermobalance . The measurements were made in a helium flow in the temperature range of 30–450 °C using the heating rate of 10 °C minXRD analysis of polycrystals was performed on a Shimadzu XRD-7000 diffractometer . Plotting of PXRD patterns and data treatment was performed using X’Pert Plus software see .1–3, we used the same “straightforward” approach 2− anions. The Sn-Br bond lengths are 2.587–2.645 Å in 1, 2.563–2.627 Å in 2, and 2.563–2.684 Å in 3, respectively. The composition of the polyhalogeno-anionic part in all cases is identical: {[SnBr6](I2)}. The I···I bonds in 1–3 are 2.692–2.695, 2.703, and 2.686 Å, respectively.The XRD data indicate that, like in halotellurates(IV), all isolated complexes are built out of mononuclear 2− units form non-covalent interactions with I2 } chains can be considered as a pseudo-2D substructure (6]2− over two closed positions (0.92/0.08 occupancies) was found in the crystal structure of 1 . The. The1, ttructure . The posure of 1 . Such cotopology . Such latopology  with shid layers . There ad layers : the fir2 and 3, I2 and [SnBr6]2− structural units form linear supramolecular chains 2[SnBr6]). Both 1 and 2 are stable and they were isolated as single phases, as follows from PXRD data .1 and 2 demonstrate different thermal stability—decomposition corresponding to continuous loss of diiodine occurs at >110 and >90 °C, respectively , Te(IV), and Sb(V) complexes, as well as dichloro-chlorostannates(IV) and –plumbates(IV) , works v


Different carbon substrates altered the morphology and alloy of Pt3Ni nanoparticles, with octahedral morphology and alloy metal preferentially developing on Ketjen black and Graphene, while spherical shape and bimetallic metal preferentially developing on Vulcan. Furthermore, the shape was shown to be regulated throughout the reduction process, with the H2:CO ratio playing a crucial role in controlling octahedral morphology and carrying out the ORR activity. At a 1:3 H2:CO ratio, the Pt3Ni/Ketjen black exhibited the highest ORR activity for both mass activity (1.02 A mgPt−1) and specific activity (5.09 mA cm−2) that were 16.5 and 66.1 times larger than commercial Pt/C catalysts, respectively (0.062 A mgPt−1 and 0.077 mA cm−2). The best ORR activity of Pt3Ni onto Graphene and Vulcan XC-72R was exhibited with a 1:1 H2:CO mixture. The catalysts were tested using a 4000-voltage-cycle accelerated durability test, and the Pt3Ni/Ketjen catalyst fared the best in terms of ORR stability and durability.The gas phase synthesis of octahedral Pt Platinum nanoparticles are the most general electrocatalysts for PEMFCs because to their high catalytic activity9. The ORR for platinum nanoparticles is commonly referred to as a sluggish kinetic, and it is the most important hindrance to the energy conversion efficiency of state-of-the-art PEMFCs15. As a result, electrocatalytic research continues to focus on ORR enhancement.PEMFCs (polymer electrolyte membrane fuel cells) are a type of fuel cells that has received a lot of attention in recent years. Electrocatalysts, which assist the oxygen reduction reaction (ORR) and hydrogen oxidation reaction (HOR) at the cathode and anode, respectively, are a vital component of PEMFC25. Using density functional theory (DFT) calculations and surface-sensitive examination, Stamenkovic et al.26 theoretically reported that the Pt3Ni in single crystal surface with (111) facet has an excellently high ORR catalytic activities, which is 10 times higher than Pt (111) surface and 90 times higher than Pt/C commercial catalyst. Following that, several studies have been carried out and published on the synthesis process of platinum-nickel nanoparticles with (111) facet or octahedral shape , which demonstrate excellent ORR catalytic activity41. The synthesis process has been classified primarily into two categories: liquid phase synthesis 41 and gas phase synthesis 33.It has been reported that the addition of other metals (M) onto Pt as Pt-M nanoparticles catalysts has been shown to boost ORR activity. Nickel (Ni) is one of these metals that is commonly employed because it is affordable, easy to get, and has good electrochemical propertiesxNi catalyst. On the other hand, a scalable, less complicate, and low-cost technique for mass manufacture of octahedral PtxNi alloy nanoparticles has been established using gas-phase synthesis33. Furthermore, this technique makes it easier to manage the atomic composition of metals and is a good way to minimize metal particle aggregation42. Colloidal PtxNi nanocrystals have intrinsic limitations due to their ligand-covered surfaces, which not only restrict free access to surface active sites but also obstruct electron transport between the catalyst and support, lowering total ORR performance. Growing nanoparticles directly on carbon supports is an effective and straightforward synthetic way to overcome this problem43. Although carbon black such as Vulcan XC-72R has been commonly employed as a support, different carbon blacks/carbons with higher specific surface area (such as Ketjen black) and superior electrical conductivity (such as Graphene) may increase ORR electrocatalytic activity. These carbon supports will impact the octahedral formation and particle size related to the H2 and CO ratio due to their distinct surface characteristics, leading the homogeneity between Pt and Ni to become either alloy or bimetallic catalysts, which has never been reported previously.Liquid-phase technique generally utilizes organic surfactants to regulate the octahedral shape. Organic surfactants commonly contaminate the catalyst surface, necessitating additional removal operations that drive up production costs and make this technique more complex and financially uncompetitive. Furthermore, liquid-phase synthesis has a poor scaling-up capacity, making it unsuitable for large-scale manufacture of the octahedral Pt3Ni nanoparticles supported on several carbon substrates . To regulate octahedral form and particle size, H2 and CO mixed gases with a Pt:Ni atomic ratio of 3:1 were examined. Transmission electron microscopy (TEM) was used to disclose the physical structure as well as the line scanning with energy dispersive X-ray (EDX) analysis. The X-Ray diffraction (XRD) analysis evaluated the alloy and bimetallic metal between Pt and Ni. In addition, the ORR activity related to specific activity (SA) and mass activity (MA) was estimated by electrochemical analysis. The accelerated durability test (ADT) was carried out in saturated oxygen up to 4000 cycles.In this work, we used a gas phase synthesis to create octahedral Pt3Ni catalysts on carbon. First, the metal precursors of platinum (II) acetylacetonate (Pt(acac)2, 48.0% of Pt, Alfa Aesar) and nickel (II) acetylacetonate (Ni(acac)2, 95%, Sigma-Aldrich) were dissolved in acetone  with molecular ratio of Pt:Ni at 3:1. Ketjen black (Cabot), Graphene (Cheap Tubes), and Vulcan XC-72R (Cabot) carbon supports were added to the solution and shaken for one hour. The acetone was then evaporated, leaving a dry combination of metals impregnated on carbons. For 1 h at 200 °C, the reduction was carried out in a combination of hydrogen  and carbon monoxide . By altering the gases volumetric flow rate, the effect of H2:CO composition on Pt3Ni nanoparticles was investigated. The overall flow rate of H2:CO was in the range of 100 to 140 cm3 min−1.Pt and Ni precursors were impregnated carbon supports, and then the reduction was carried out in the gas phase, resulting in octahedral Pt3Ni nanoparticles on various carbons were observed by transmission electron microscopy . The atomic percentage and the elemental distributions were characterized by energy dispersive spectroscopy which operated on TEM . The crystalline structure of prepared nanoparticles was analyzed using X-ray diffraction analysis .The specifics surface area of carbon supports was examined by the brunauer–emmett–teller (BET) analysis. The inductively coupled plasma-atomic emission spectrometry  was used to measure the metal loadings of Pt and Ni. The morphologies and dispersion of Pt3Ni/C electrocatalysts were evaluated by the voltammetry technique via a standard three-electrode system at room temperature using a bi-potentiostat (Pine Instrument Co.). A glassy carbon of the rotating disk (GC-RDE) was a working electrode , whereby a reference electrode was a single junction silver chloride electrode  in 3 M KCl. A counter electrode was a platinum wire located nearby the working electrode. The catalysts inks were prepared using 10 mg of Pt3Ni/C mixed in 5 ml of stock solutions, which properly composed of deionized water (RCI Labscan), isopropanol (C3H8O (IPA), RCI Labscan), and Nafion solution . The catalysts inks were sonicated for well dispersion for 20 min, and then, carefully dropped onto GC-RDE (i.e. working electrode), which had physical surface area of 0.19625 cm2. The Pt loading on GC-RDE were kept constant around 40 µgPt cm−2. The cyclic voltammetry (CV) was applied to evaluate the hydrogen under potential deposition (HUPD), which was a measurement of the electrochemical active surface areas (ECSA). The CVs were carried on under He-saturated in 0.1 M perchloric acid  as the supporting electrolyte at a scanning rate of 50 mV s−1 at room temperature. The linear sweep voltammetry (LSV) technique was used for measuring the oxygen reduction reaction (ORR) of catalytic activities. The LSV was carried out under O2-saturated in 0.1 M HClO4 electrolyte at a scanning rate of 20 mV s−1 with a rotation speed of 1600 rpm. The electrode was activated by CV for 40 cycles prior to LSV. The mass activity (MA) and specific activity (SA) of electrocatalysts were calculated using kinetics current (ik). The polarizations of ORR were corrected with the iRsoln drop, and the background (b.g.) currents were also corrected according to the Ref.9. Furthermore, the ORR stability was carried on via the accelerated durability test (ADT) by running the CV and LSV up to 4000 cycles (about 22 h. for each sample). The CVs were operated in the voltage range from 0.4 to 1.2 VSHE at a scanning rate of 50 mV s−1. The LSVs were operated at a scanning rate of 20 mV s−1 from 0.2 to 1.2 VSHE. In addition, the electrochemical properties of commercial Pt/C catalysts  were compared with the same conditions.The electrochemical properties of prepared Pt2 g−1, followed by Graphene and Vulcan XC-72R with 887.37 m2 g−1 and 259.08 m2 g−1, respectively. ICP was used to measure the mass loadings of Pt and Ni during impregnation (Table Ketjen black (K), Graphene (G), and Vulcan XC-72R (V) were the three types of supported carbons studied. According to BET study, Ketjen black has the largest specific surface area with 1499.11 m2 and CO environments with volume flow rates (cm3 min−1) ranging from 100:0 (pure H2) to 60:60 (1:1), 30:90 (1:3), 20:120 (1:6), 10:120 (1:12), 5:120 (1:24) and 0:100 (pure CO). As shown in Fig. 3Ni nanoparticles with varying H2:CO ratios were photographed by TEM at the same magnification of 400,000 times. Figure 3Ni/K electrocatalysts, whereas Fig. 3Ni/G and Pt3Ni/V electrocatalysts, respectively. For all supported carbons, when the reducing gas was pure H2, as shown in Fig. 3Ni congregated near to one other and probably agglomerated to form the lump of Pt metals, especially in Graphene support. As soon as the CO was mixed into the reducing gas, the Pt3Ni particles were well dispersed with various shapes and sizes at particular H2:CO ratio. Pt3Ni average particle sizes from TEM images on Ketjen black, Graphene, and Vulcan supports with varied H2:CO ratios are shown in Fig. 3Ni particles on Ketjen black had the highest size, regardless of the H2:CO ratio, whereas those on Graphene and Vulcan were smaller and smaller, respectively. This might be related to the specific surface area of supports, which, as indicated in Table 45. Furthermore, the results demonstrate that as CO content grew until pure CO was reached, Pt3Ni particle size in all supports did reduce.All impregnated carbons were reduced in H cm3 min− ranging  cm3 min− ranging  cm3 min− ranging  cm3 min− ranging  cm3 min− ranging  cm3 min− ranging 3Ni particles changed considerably when the H2:CO ratios were changed. According to the TEM pictures, the nanoparticles of Pt3Ni/K and Pt3Ni/G had the edge corner and were most likely rhombus-shaped. As a result of this, the octahedral morphology developed46. The octahedral Pt3Ni/K electrocatalyst was formed in the H2:CO compositions ranging from 1:3 to 1:24, as illustrated in Fig. 3Ni/G electrocatalyst was produced in H2:CO ratios ranging from 1:1 to 1:24, as seen in Fig. 3Ni/V was not fully formed, and most of them had a round corner, resulting in a typical spherical shape. As a result, the types of supported carbon have a substantial influence on the shape of Pt3Ni metal.The formation of Pt2:CO ratio is one of the most essential factors in controlling particle shape and size, particularly in solid-state techniques The adsorption of CO on Ni and Pt metals has long been recognized to cause octahedral formation27. As shown by the CO adsorption in the Pt-Ni precursor, Ni tends to develop as Ni (111), while Pt prefers to develop as Pt (100)48. Meanwhile, H2 aids in the migration of both metal precursors (Ni and Pt) onto carbon supports and their subsequent reduction into alloys46. In comparison to H2/CO flowing gases, the particle size of Pt3Ni with flowing pure CO was smaller, and the morphology resembled a blend of octahedron and polyhedron as shown in Fig. 2 was added during reduction, the particle sizes increased considerably and the morphology seemed to be spherical owing to the absence of CO. Furthermore, the formation of polyhedral or spherical shapes on Vulcan substrate might be caused by a restricted supply of metal precursors, especially Ni, to growth sites, as well as inadequate migration. This indicates that the 3:1 atomic ratio of Pt to Ni is insufficient for the Vulcan carbon-forming octahedron.Furthermore, the Hop as Ni 11, while3Ni/Ketjen black, Pt3Ni/Graphene, and Pt3Ni/Vulcan XC-72R were plotted as shown in Fig. 2:CO compositions in the ratios of 1:1, 1:3, 1:6, 1:12, and 1:24 were studied. The free kinetic current  and specific activity (SA) were calculated using Eqs. (51.Pt−1) of the electrocatalyst and −2) of the electrocatalyst and ECSA is the electrochemical active surface area of the catalyst measured by hydrogen under potential deposition (HUPD) method.The linear sweep voltammograms (LSV) were used to determine the electrocatalytic activity of the ORR. After normalization and electrolyte resistance adjustment, the LSV results for Pt3Ni electrocatalysts on Ketjen black, Graphene, and Vulcan XC-72R are shown in Fig. 2:CO ratio. It was discovered that Pt3Ni electrocatalysts on various carbon supports exhibited distinct maximal ORR activity at various H2:CO ratios, with Ketjen black having a 1:3 ratio and Graphene and Vulcan XC-72R having a 1:1 ratio. Clearly, Pt3Ni onto Ketjen black (with an H2:CO ratio of 1:3) had the highest ORR catalytic activity, with MA of around 1.02 A mgPt−1 and SA of approximately 5.09 mA cm−2, as shown in Fig. 3Ni particles were completely dispersed onto carbon supports and clearly revealed an octahedral form for Pt3Ni/K (1:3) and Pt3Ni/G (1:1). On the other hand, Pt3Ni/V (1:1) particles were generally spherical in form and had a particle size as tiny as 3.7 nm. According to C. Zhang et al.28, the octahedral Pt3Ni electrocatalysts should have a particle size of around 6 to 10 nm. As a result, it is once again proven that Pt3Ni/V (1:1) was unlikely to have an octahedral structure.The mass and specific activity of Pt3Ni nanoparticles, but also the ORR electrocatalytic activities. The ORR activities of Pt3Ni nanoparticles on various carbon supports were optimum at distinct H2:CO ratios. For this reason, the H2:CO ratios that are optimal for each carbon support to produce the Pt3Ni/C electrocatalyst should be taken into consideration. The following section discusses in further detail the comparison of Pt3Ni nanoparticles on each carbon support that had the highest ORR catalytic activity.Results show that carbon supports not only affect the physical characteristics of Pt3Ni/K (1:3), Pt3Ni/G (1:1), and Pt3Ni/V (1:1)) were further determined. The high-resolution TEM pictures on individual particles in Fig. 3Ni/K (1:3) and Pt3Ni/G (1:1) was clearly octahedron, while Pt3Ni/V (1:1) was spherical. These three photos showed a lattice spacing of 0.22 nm, indicating the surface of Pt-Ni nanoparticles with (111) facets 32. Furthermore, XRD analysis of the powders of those catalysts revealed that they had a face-centered cubic (fcc) structure, as shown in Fig. 3Ni/K (1:3) and Pt3Ni/G (1:1) to the Ni (111) peak indicates the creation of a Pt-Ni alloy. On the other hand, the Pt (111) diffraction peak of Pt3Ni/V (1:1) did not shift to Ni (111), but the spectrum seemed to split into two peaks, one for Pt (111) and another for Ni (111). This indicates that Pt and Ni were only weakly bound and did not form the Pt-Ni alloy but rather formed a bimetallic structure.The three samples with the highest ORR activity , Pt3Ni/G (1:1), and Pt3Ni/V (1:1) were compared to those of a commercial Pt/C catalyst. The cyclic voltammograms (CV), Fig. −1, and the electrochemical active surface area (ECSA) was derived from the hydrogen under potential deposition (HUPD) using the following equation50.As shown in Fig. 2 gPt−1) is the electrochemical active surface area of electrocatalysts, −2) is a charge density conversion factor during hydrogen adsorption–desorption area of Pt polycrystalline.The ECSA , Pt3Ni/G (10.45 m2 gPt−1), and Pt3Ni/V (8.23 m2 gPt−1). It was also found that the Pt3Ni/K had the largest double layer, followed by Pt3Ni/G, commercial Pt/C, and Pt3Ni/V, respectively. The double layer was different because of the different structures of the carbon supports that were used. This was seen in a double layer comparison between Pt3Ni/V and commercial Pt/C, where the carbon support was the same Vulcan XC-72R. The likelihood for double layer formation was shown to be connected to the specific surface area of carbon supports, with Ketjen black having the greatest specific surface area (1499.11 m2 g−1) and the biggest double layer.Figure 3Ni/K (1:3) catalyst had the greatest MA of 1.02 A mgPt−1, followed by the Pt3Ni/G (1:1) and the Pt3Ni/V (1:1) catalysts, and that the commercial Pt/C catalysts had the lowest MA of 0.062 A mgPt−1, as shown in Fig. 3Ni/K (1:3) yielded the greatest value of 5.09 mA cm−2, which was 1.5 times higher than Pt3Ni/G (1:1), 1.8 times higher than Pt3Ni/V, and 66.1 times higher than commercial Pt/C catalyst.The ORR electrocatalytic activity of the pristine samples (after 40 cycles of activation) was investigated using the LSV, as shown in Fig. 3Ni/C samples had much higher ORR electrocatalytic activity for MA and SA than commercial Pt/C catalysts, despite the commercial catalysts had the greatest ECSA. This is most likely owing to the fact that the electronic structure of Pt is altered when Ni is added, resulting in the formation of a Pt-Ni alloy or bi-metal. This enhances oxygen molecule binding onto the metal surface42. Additionally, the formation of an octahedral shape, such as the (111) plane in a Pt-Ni alloy, had a significant effect on the ORR activity52. Thus, Pt3Ni/K (1:3) and Pt3Ni/G (1:1) demonstrated much higher ORR activity (both MA and SA) than Pt3Ni/V (1:1), which had a spherical form and was not an alloy catalyst .The results indicate that all Pt3Ni/K (1:3) retained the greatest MA and SA values of 0.24 A mgPt−1 and 1.17 mA cm−2, respectively. On the other hand, commercial Pt/C catalysts produced the least MA (0.004 A mgPt−1) and SA (0.005 mA cm−2). According to the results between pristine and after ATD, the ORR activity losses were evaluated and plotted on the right-hand side scale of Fig. 3Ni/K (1:3) loses around 76.9 percent of its activity, whereas Pt3Ni/G (1:1) and Pt3Ni/V (1:1) lose approximately 95.3 percent and 91.5 percent of their activity, respectively. This indicates that Pt3Ni/K (1:3) had the greatest stability and durability, followed by Pt3Ni/V (1:1) and Pt3Ni/G (1:1).SA and MA were computed after 4,000 cycles and are displayed in the right-hand histograms of Fig. 54 and the ORR activity was deteriorated as a result. In Fig. 3Ni/K (1:3) seemed to have a less collapsed octahedral form than Pt3Ni/G. (1:1). Furthermore, the Ni leached away the most in the Pt3Ni/V (1:1) sample, resulting in lost bimetal behavior and dramatically reduced ORR activity. As a result, it verifies once again that Pt3Ni/K (1:3) exhibited the greatest stability and durability of these samples.The elemental mapping images and line scanning profiles in Fig. 3Ni/C electrocatalyst for ORR at the cathode side of PEM fuel cells. Regardless of H2:CO composition, the Pt3Ni on Ketjen black had the highest particle size, followed by Graphene and Vulcan XC-72R, which corresponded to specific surface area. On Ketjen black and Graphene, octahedral Pt3Ni nanoparticles with alloy behavior were clearly detected, whereas spherical particles with bimetallic behavior were identified on Vulcan XC-72R. The ORR activities of Pt3Ni nanoparticles on each carbon support were best at distinct H2:CO ratios, which were 1:3 for Pt3Ni/K and 1:1 for Pt3Ni/G and Pt3Ni/V. The ORR activity of Pt3Ni/K (1:3) was the greatest, followed by Pt3Ni/G (1:1) and Pt3Ni/V (1:1). These Pt3Ni /C, on the other hand, lose their ORR activity after ADT voltage cycles. Their stability is harmed by the collapse of the octahedral structure. As a result, additional efforts to improve stability will be required. Doping or infusing the third or fourth metals is likely to prevent the octahedral structure from collapsing.The gas phase synthesis was shown to be an effective method for producing an octahedral PtSupplementary Information.


Although biotechnology is considered as an environmentally friendly method to produce chemicals, almost all biochemicals face carbon dioxide emission from inevitable respiration and energy metabolism of most microorganisms. To cater for the broad prospect of biochemicals, bioprocess optimization of diverse valuable products is becoming increasingly important for environmental sustainability and cleaner production. Based on Ca(OH)2 to CO2 in exhaust gas showed a remarkable 152-fold increase in the 5 M Ca(OH)2 group compared to 5 M NaOH as the CO2 capture agent. Green hydrogen in exhaust gas ranged between 17.2% and 20.2%, with the remainder being N2 with negligible CO2 emissions. During CO2 capture in situ, micro-nano calcite particles of CaCO3 with sizes in the range of 300 nm to 20 µm were formed simultaneously. Moreover, when compared with 5M NaOH group, the concentrations of soluble salts and proteins in the fermentation broth of 5 M Ca(OH)2 group were notably reduced by 53.6% and 44.1%, respectively. The remarkable reduction of soluble salts and proteins would contribute to the separation of 1,3-PDO.In fed-batch fermentation, the maximum concentration of 1,3-PDO reached up to 88.6 g/L with an overall productivity of 5.54 g/L/h. This productivity is 31.9% higher than the highest value previously reports (4.20 g/L/h). In addition, the ratio of H2 was used as a CO2 capture agent and pH regulator in this study to promote the production of 1,3-PDO. Meanwhile, micro-nano CaCO3 and green H2 were co-produced. In addition, the soluble salts and proteins in the fermentation broth were significantly reduced.Ca(OH)The online version contains supplementary material available at 10.1186/s13068-022-02190-2. More recently, some proposals for biochemicals production by researchers have focused on optimizing processes for environmental sustainability, identifying cost-effective [2 into valuable products [2 is becoming essential.There is a global consensus on the urgent need to reduce carbon footprints to develop a renewable economy and protect our habitable planet. For more than 40,000 years prior to 1912, the atmospheric COCrude glycerol is a primary by-product of the oleochemical industry, such as biodiesel, soap, fatty acid, and fatty ester production . For eveKlebsiella pneumoniae, Clostridium butyricum and Lactobacillus reuteri and so on [C. butyricum is considered as one of the most excellent 1,3-PDO producers due to its superior performance in efficient 1,3-PDO production and less by-products. Meanwhile, it is suitable for industrial production of 1,3-PDO, because it is a probiotic in the intestinal tract and B12-independent bacterium [C. butyricum DL07 could produce 104.8 g/L 1,3-PDO from glycerol, and the productivity was up to 3.38 g/L/h in fed-batch fermentation [Glycerol, as a substrate, could be converted into many value-added chemicals, such as 1,3-propanediol , 2,3-butanediol, citric acid, n-butanol, ethanol, lipids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), using a variety of microorganisms via different metabolic pathways , 20–26. nd so on –32. C. bacterium . In the entation , which aC. butyricum has been well-recognized by intracellular metabolic analysis [C. butyricum. Otherwise, the organic acids could inhibit 1,3-PDO production, because more energy is needed to maintain the pH in the cell [+, K+ and NH4+ from the alkalis are introduced with high concentration into the fermentation broth, which cause high osmotic pressure and inhibiting the growth of bacterial cells [2 and H2 are produced in the oxidation branch of glycerol metabolism [2 not only puts pressure on the environment but also reduces carbon utilization. In addition, the dissolution of CO2 in fermentation broth would require more alkaline solution to neutralize the fermentation pH. Then, the large amounts of soluble salt in the fermentation broth bring difficulty in the separation of 1,3-PDO. Therefore, the recovery of CO2 and the reduction of soluble salt concentration in fermentation broth are expected in the microbial production and separation of 1,3-PDO. At the same time, H2 with a relatively high purity will be available as a clean, sustainable, and ideal energy resource.The intracellular metabolic pathway of glycerol in analysis . 1,3-PDOanalysis . Usuallythe cell , 37. In the cell , 38, 39.al cells . Furthertabolism . The for3), an important material, have been widely used in many fields, such as the construction industry, paper industry, cosmetics, toothpastes, water treatment, pigments, and drug delivery systems [3 has attracted many researchers’ interests in recent years because of its good properties, such as the high ratio of surface area to volume, high porosity, non-toxicity and compatibility toward the human body [3 can exist in three crystal forms: calcite, vaterite, and aragonite. Calcite is the most stable polymorph and one of the most common minerals on Earth, serving as the main component of sedimentary limestone. At present, calcite (CaCO3) could be synthesized by chemical method and microbially induced precipitation (MIP). MIP is more favored by researchers over chemical synthesis, because it can obtain the controlled single crystal [3 usually takes several days and is only produced in small quantities. In a report, Rhodococcus degradans BaTD-248 was cultured for 3 and 21 days to produce CaCO3 [3 [Nano- and micro-particles of calcium carbonate 2 and double CO2 capture agents (NaOH & Ca(OH)2) will act as CO2 capture agents and pH regulators for 1,3-PDO production. The various Ca(OH)2 concentrations and stirring speeds were investigated to produce 1,3-PDO, H2, and micro-nano CaCO3. Moreover, the effects of this process on the salt concentration, ionic composition and soluble protein concentration in the fermentation broth were discussed.The aim of this study is to propose a novel integrated process for the production of multiple products while avoiding CO2 as exhaust gas, due to redox homeostasis and energy balance during the glycerol metabolism [2 capture agent and pH regulator, NaOH was selected to absorb CO2 and neutralize the organic acids, e.g., 5 M NaOH solution used for anaerobic fermentations by C. butyricum DL07. To create anaerobic fermentation environment, nitrogen gas was bubbled into the fermentation medium for 1 h before and after inoculation, respectively, or continuously throughout the whole fermentation process. Regardless of the conditions in the preceding two cases, efficient 1,3-PDO production could be achieved, as shown in Fig. 2 captured in the two cases.During the microbial fermentation of 1,3-PDO, organic acids were produced as by-products, as well as COtabolism , 49, 50.C. butyricum IK 124 produced 80.1 g/L of 1,3-PDO with a productivity of 1.80 g/L/h in a fed-batch fermentation using crude glycerol as the substrate [K. pneumoniae DSM 4799 from crude glycerol [C. butyricum DL07 could achieve higher concentration  and productivity (2.84 or 2.85 g/L/h) in this study. When the above two flushing nitrogen gas modes were compared, they had almost no effect on the production of 1,3-PDO and by-products. About 0.340 mol/L organic acids were generated in both fermentation groups. In other words, about 216 g 5 M NaOH solution was required to neutralize the organic acids produced during fermentation, theoretically. However, the theoretical value was less than the actual consumption of 5 M NaOH solution in the first case (328 g) or the second one (241 g), implying that CO2 fixation levels differed, i.e., 0.24 mol CO2 fixed in the first case vs. 0.05 mol CO2 fixed in the second one. This demonstrated that the flushing time of nitrogen gas could affect the fixation of CO2. Shorter flushing time, more CO2 would be fixed into Na2CO3. Unfortunately, a large amount of Na2CO3 in the fermentation broths poses a significant challenge for the separation of 1,3-PDO. Indeed, it is not ignored that a large amount of exhaust gas composed of CO2 and H2 from fermentation is discharged into the atmosphere. Therefore, a more significant reduction in CO2 emissions and soluble salts in the fermentation broth is essential for the industrial fermentation of 1,3-PDO.ubstrate . Similarglycerol . In cont2 as a CO2 capture agent and pH regulator, fed-batch fermentations were performed with different concentrations of Ca(OH)2 suspension in comparison to 5 M NaOH solution, as shown in Table 2 suspension was used, the concentration and productivity of 1,3-PDO were higher, i.e., 85.1 vs. 88.6 g/L and 2.84 vs. 5.54 g/L/h, respectively, when compared to the fermentation using 5 M NaOH, as shown in Fig. 2 suspension decreased, the concentration and productivity of 1,3-PDO were also reduced. For example, only 76.3 g/L 1,3-PDO was produced if Ca(OH)2 concentration decreased to 1.5 M, which is mainly attributed to a dilution effect of low alkaline concentration on fermentation broth. The concentration of butyric acid and acetic acid dropped moderately as a decline of Ca(OH)2 concentration. Unexpectedly, lactic acid was generated using Ca(OH)2 about twice higher than NaOH as listed in Table 2 group. On the other hand, stirring speed between 150 and 350 rpm had little effect on fermentation, indicating adequate mixture homogeneity of the fermentation broth obtained at the given stirring speeds and no other effects on microbial metabolism [To verify the feasibility of 1,3-PDO and other organic acids production using Ca(OH)tabolism .Table 2F2 suspension was surprisingly 31.9% higher than the previously reported highest level (4.20 g/L/h) of fed-batch fermentation and reached the productivity of continuous fermentation [Up to date, the highest concentration of 1,3-PDO produced by natural producers was 104.8 g/L with a productivity of 3.38 g/L/h, which was achieved in fed-batch fermentation using pure glycerol and a large amount of yeast extract in our previous study . In contentation , 54. Morentation .2 than NaOH (30 h). A comparison of Fig. 2 group, whereas a significant reduction occurred at the end of fermentation in the 5 M NaOH group. Despite the fact that adding a larger amount of Ca(OH)2 suspension dilutes the fermentation broth, the total 1,3-PDO concentration and productivity achieved in the Ca(OH)2 group were higher than in the NaOH group. This might be due to both Ca2+ stimulation on cell growth and a decrease in osmotic pressure, which is relevant to adding Ca(OH)2 during fermentation. Undoubtedly, a high osmotic pressure caused by high soluble salt concentration, e.g., NaOH group, poses a challenge to cell survival and metabolism [2 group will be discussed in Sect. 2 group compared to the 5 M NaOH group. It was reported that the presence of Ca2+ could contribute to the formation of lactic acid [43− in the fermentation, which will be discussed in detail in Sect. Desalination and deprotein of the fermentation brothIt should be emphasized that such high productivity and concentration were achieved in a much shorter fermentation period 16 h) using Ca(OH)6 h usingtabolism . The destic acid , 57. In 2 group, it was comparable to the reported yield of K. peneumoniae. For example, a yield of 0.454 g /g with 80.2 g/L 1,3-PDO was obtained by K. peneumoniae DSM4799 from crude glycerol [K. peneumoniae LX3 obtained a yield of 0.487 g/g accompanied by 71.4 g/L 1,3-PDO using refined glycerol as the substrate [K. peneumoniae, the production of 1,3-PDO by C. butyricum DL07 was still competitive using Ca(OH)2 in the fermentation because of the high concentration of 1,3-PDO (88.6 g/L) with a considerable productivity (5.54 g/L/h) from crude glycerol. Moreover, the CO2 produced in the fermentation was captured in large quantities, resulting in the insoluble CaCO3 in the fermentation broths. This process is environment-friendly while relieving the pressure of 1,3-PDO separation. Specific details will be discussed later.Although the yield of 1,3-PDO (0.481–0.486 g/g) was reduced in the Ca(OH)glycerol , and K. ubstrate . In cont2 capture agents was attempted for the production of 1,3-PDO, aiming at increasing the 1,3-PDO yield and reducing the soluble salt concentrations in the fermentation broth. In the first 12 h of fermentation, 5 M NaOH was used to capture CO2 as well as to regulate the fermentation pH, and then 5 M Ca(OH)2 suspension instead of 5 M NaOH was employed to proceed with the fermentation. The results showed that the concentration of 1,3-PDO was 84.4 g/L in 17 h, accompanied by a productivity of 4.96 g/L/h 2 or NaOH & Ca(OH)2), C. butyricum DL07 could produce up to 84.4–88.6 g/L 1,3-PDO with the yield of 0.481–0.506 g 1,3-PDO/g glycerol. Moreover, the 1,3-PDO productivity was about 2.84–5.54 g/L/h. These values indicated that C. butyricum DL07 had excellent 1,3-PDO production performance according to 1,3-PDO production using different pH regulators in some recent reports 2 group, while butyric acid (14.2 g/L) and acetic acid (9.81 g/L) showed negligible changes. The production of lactic acid was only 1.17 g/L, which was similar to that of NaOH group, but greatly different from the Ca(OH)2 group 2).A two-stage pH control strategy using double pH regulators has been explored to produce docosahexaenoic acid . The comric acid . RegardlC. butyricum DL07, when Ca(OH)2 was the only CO2 capture agent and pH regulator, the productivity of 1,3-PDO was greatly improved. Regrettably, the yield of 1,3-PDO has decreased. A process with satisfactory 1,3-PDO yield, productivity and concentration is expected to reduce 1,3-PDO production costs. Fortunately, using double CO2 capture agents resulted in an increase in 1,3-PDO yield (0.506 g/g) with satisfactory 1,3-PDO concentration (84.4 g/L) and productivity (4.96 g/L/h). Furthermore, the production of lactic acid showed the same trend  in the double CO2 capture agents group as compared to the NaOH group, whereas more lactic acid was produced after 5 h of fermentation in Ca(OH)2 group 2 acts as a CO2 capture agent allowing for cleaner production and the synthesis of valuable products. In theory, CaCO3 could be generated in fermentation once Ca(OH)2 reacts with CO2. The concentration of Ca(OH)2 suspension and the stirring speed of fermentation have a non-negligible effect on the size of CaCO3 particles [3 was investigated using various Ca(OH)2 concentrations and fermentation stirring speeds  was obtained in fermentation. The precipitates showed a decreasing trend as the Ca(OH)2 concentration was reduced. Subsequently, the precipitations collected from fermentations were observed by SEM. Most particles with a size of 10–15 μm were produced using 5 M Ca(OH)2 suspension and the stirring speed of 250 rpm. When the concentration of Ca(OH)2 suspension was reduced to 2.5 M, a large proportion of particles were distributed between 5 and 10 μm. Using 1.5 M Ca(OH)2 suspension as the CO2 capture agent, particles with a large size limit of 5 μm were obtained. In addition, by increasing the fermentation stirring speed to 350 rpm, further small-size precipitations were observed. Precipitations in fermentation were smaller than 5 μm in the 2.5 M Ca(OH)2 group. Surprisingly, the nano-particles were produced using 1.5 M Ca(OH)2 suspension. The precipitations were not completely individual particles, according to the SEM images, but a portion of them were aggregated.Harticles . As a re2 group was presented in Fig. 3 was also conducted to serve as standard sample. As a result, calcium, carbon, and oxygen as the most abundant elements were determined in precipitations collected in the fermentation. Further comparison with the EDX spectra of pure CaCO3, revealed a high degree of similarity in the EDX spectra between these precipitations and pure CaCO3. it can be concluded that the precipitations produced during fermentation were CaCO3 particles. Finally, the crystal form of CaCO3 particles was characterized by XRD. As indicated by XRD spectra  at 15.0 keV to identify the elemental compositions in the precipitations. The EDX spectra for the precipitations in 5 M Ca(OH)Bacillus pasteurii, Bacillus mucilaginosus, Bacillus alcalophilus, Photosynthetic bacteria could induce CaCO3 formation except C. butyricum [2 released by C. butyricum could undoubtedly react with Ca(OH)2, resulting in the formation of CaCO3. The precipitations of various sizes in the fermentation were observed by a SEM, while the concentration of Ca(OH)2 and the stirring speed of the fermentation were changed. Obviously, the size of CaCO3 particles formed in fermentation decreased as the concentration of Ca(OH)2 suspension decreased and the stirring speed of fermentation increased. After all, the stirring speed can affect the precipitation crystal and size [2 suspension with 250–350 rpm stirring speed as well as 1.5M Ca(OH)2 suspension with 250 rpm stirring speed. The nano-particles were synthesized using the 1.5 M Ca(OH)2 suspension and a stirring speed of 350 rpm. Therefore, it can be concluded that micro/nano CaCO3 was produced as calcite during the production of 1,3-PDO by C. butyricum DL07. Bacteria could induce various crystalline CaCO3 in the form of calcite, aragonite, vaterite and amorphous CaCO3. In general, the effects of induction conditions and time on the crystal form and size of CaCO3 are investigated using microbial induction method, but the productivity of CaCO3 is rarely reported. Magnesium, potassium, phosphate, citric acid, amino acids, polysaccharides, and proteins have all been linked to the formation of CaCO3 crystals [C. butyricum DL07. A complex process resulted in the production of stable calcite (CaCO3), allowing CO2 to be converted into high value products during the fermentation. Although calcite is the most common crystalline form, the time required to form CaCO3 was reduced from more than 3 days reported by microbial induced method to 16 h using glycerol fermentation [3 and C. butyricum could be obtained separately by differential centrifugation after fermentation because of their different sedimentation coefficients. Calcite CaCO3 could be widely applied in the construction industry. Moreover, a popular mixture of CaCO3 and living probiotics can be widely used as animal feed.It has been reported that many microorganisms, including utyricum , 71, 72.and size , 74. Thecrystals –48. It centation . To our 2 and CO2 as exhaust gas was released from most microbial fermentations into the environment [2 as CO2 capture agent, the composition of exhaust gas was detected and expressed as percentage difference 2 group, 28.6 in the 2.5 M Ca(OH)2 group, and 36.2 in the 1.5 M Ca(OH)2 group. Regrettably, in the double CO2 capture agents group, the ratio of H2 to CO2 was reduced to 0.203, with a H2 proportion of 9.21% and CO2 proportion of 45.3% in exhaust gas. The highest H2 production of 15.9 mmol/L of medium was achieved using 5 M Ca(OH)2 suspension and 11.0 mmol/L of medium as the lowest concentration was produced with 1.5 M Ca(OH)2 suspension as the CO2 capture agent.In general, a mixture of Hironment , 76. Basnce Fig. , with 5 C. butyricum is commonly employed to produce H2 [2 to CO2 in exhaust gas of fermentation would increase if CO2 was captured, which will contribute to the separation of H2. Expectedly, the proportion of CO2 significantly decreased, because CO2 was converted into CaCO3 particles in the fermentation. Indeed, there was a significant reduction in the proportion of CO2 (< 1.75%) in the 5 M Ca(OH)2 group. The results showed that the ratio of H2 to CO2 was 152 times higher in the 5 M Ca(OH)2 group than the 5 M NaOH group indicating that Ca(OH)2 had a more outstanding CO2 fixation level. As the Ca(OH)2 concentration decreased, the production of H2 and CO2 was declined, but has led to a rising ratio of H2 to CO2 2 concentration made Ca(OH)2 to fix CO2 more efficiently. Thus, the proportion of CO2 in the exhaust gas decreased as Ca(OH)2 concentration decreased, and the value was almost close to zero. On the contrary, the proportion of H2 in the exhaust gas increased slightly as Ca(OH)2 concentration decreased. Therefore, the H2/CO2 ratio decreased with Ca(OH)2 concentration significantly. Apparently, the ratio of H2 to CO2 in the double CO2 capture agents group (0.203) had a significant decrease compared to the Ca(OH)2 group due to the poor CO2 capture agent (NaOH) acting in the first 12 h of the fermentation. There is no doubt that more CO2 would be fixed to form CaCO3 in situ as long as the excellent CO2 capture agent (Ca(OH)2) was employed as soon as possible in the fermentation, surely obtaining a higher ratio of H2 to CO2. Therefore, it can be concluded that the earlier Ca(OH)2 is introduced into fermentation, the higher H2 ratio with lower CO2 ratio appears in the exhaust gas. In the future, the addition time of Ca(OH)2 should be optimized for a higher ratio of H2 to CO2 in double CO2 capture agents. The accumulated H2 production ranged between 11.0 and 15.9 mmol/L of medium, which was in agreement with the previous reported [2 production decreased as Ca(OH)2 concentration decreased, following the same trend as the production of 1,3-PDO, owing to a dilution effect of Ca(OH)2 suspension. Studies on microbial fermentation accompanied by in situ CO2 capture are rarely reported. Many studies had reported biohydrogen production using C. butyricum, but only focusing on the total hydrogen production, not on the ratio of H2 in the exhaust [2 to CO2 is concerned with the separation of H2, since normally CO2 plays an indispensable role in most microbial fermentation [2 and improves the ratio of H2 in the exhaust gas in the future.oduce H2 . The ratof CO2 < .75% in treported . H2 prod exhaust –81. Actuentation . This ne32+ was present in the fermentation broth, as CO2 produced by microbial metabolism dissolved in water. As a result, once Ca(OH)2 was selected as the CO2 capture agent rather than NaOH, insoluble CaCO3 instead of soluble Na2CO3 would present in the fermentation broth. To further reduce soluble salts in the fermentation broths, quantitative ammonium hydroxide as a pH regulator was added after fermentation initiation to substitute ammonium sulfate of the fermentation media as an inorganic nitrogen source without affecting the 1,3-PDO concentration . Thus, the salt concentration of the fermentation broth was reduced by 53.6%. The fermentation broth showed a relatively low conductivity when ammonium hydroxide replaced ammonium sulfate of the fermentation media, mainly due to reduced sulfate. Apparently, when Ca(OH)2 was added into the fermentation, the conductivity values rose slowly during the subsequent fermentation, indicating the production of insoluble salts. It demonstrated that the addition of Ca(OH)2 can effectively reduce the soluble salt concentrations of the fermentation broth, thereby contributing to the separation of 1,3-PDO. In addition, the productivity and concentration of 1,3-PDO in Ca(OH)2 group were significantly higher than in NaOH group. It is quite possible that the formation of insoluble CaCO3 would result in lower osmotic pressure of fermentation broth, which leads to a lower osmotic pressure for cell, which is beneficial for cell survival and 1,3-PDO production.The higher salt concentration in the fermentation broth could affect 1,3-PDO fermentation efficiency, because the high osmotic pressure caused by a large amount of soluble salts poses a challenge to cell survival . More im2 capture agents, when 5 M Ca(OH)2 suspension was introduced in fermentation at 5 h, the conductivity value of the fermentation broth was 20,960 μs/cm, whereas when Ca(OH)2 was involved in fermentation from 11 h, the conductivity was 23,900 μs/cm. Thus, the earlier Ca(OH)2 was involved in fermentation, the less soluble salts were present in the fermentation broth. To further clarify the main ion species and specific concentration existed in the fermentation broths, concentration of ions was determined at the end of fermentation. The results showed that large differences in concentration were exhibited in some ions  during the fermentation with different CO2 capture agents (Table + of fermentation broth was reduced by 99.7% in the 5 M Ca(OH)2 group (38.61 mg/L). Conversely, Ca2+ concentration was increased from 64.74 mg/L to 6308.75 mg/L. When Ca(OH)2 participated in the fermentation, there was no PO43− in the fermentation broth. This indicated that Ca3(PO4)2 precipitations existed in the fermentation broth. At the same time, regardless of the fermentation conditions, the NH4+ added to the media was almost absent at the end of the fermentation, which should be attributed to the microbial uptake. There was little difference in the concentration of Cl− and K+ concentrations, although different CO2 capture agents were used in the fermentation. Overall, the total ion concentration in Ca(OH)2 group was about half that of the NaOH group.For using double CO2 suspension and 5 M NaOH were selected as the CO2 capture agent and pH regulator, respectively. The soluble protein concentration of 5M Ca(OH)2 group was only 1.52 g/L, while that of the 5 M NaOH group was up to 2.72 g/L. This result suggested that the soluble proteins of the fermentation broth decreased by 44.1% with 5 M Ca(OH)2 suspension as the CO2 capture agent and pH regulator when compared to 5 M NaOH. It is speculated that the reduced salt concentrations in fermentation broth caused by insoluble CaCO3 pose fewer threats to cells, allowing for the avoidance of proteins synthesis and bacterial cell lyses. After all, high osmotic stress imposes pressure on bacteria to synthesize multiple characteristic proteins, such as membrane proteins and translocators [Surprisingly, there were also significant differences in soluble protein concentration of fermentation broth when 5 M Ca(OH)locators , and it locators . Lower sC. butyricum DL07 either using NaOH, Ca(OH)2 or both combinations as the CO2 capture agent and pH regulator. The highest 1,3-PDO concentration (88.6 g/L) accompanied by the highest reported productivity (5.54 g/L/h) was achieved, while 5 M Ca(OH)2 suspension acted as the CO2 capture agent in fed-batch fermentation. Simultaneously, CO2 was captured in situ to produce CaCO3 and improve the ratio of H2 to CO2 in the exhaust gas. Furthermore, the CaCO3 produced during fermentation existed in calcite, and its size gradually decreased as the concentration of the Ca(OH)2 suspension decreased and fermentation speed increased. Nano-CaCO3 was synthesized in 1.5 M Ca(OH)2 group with a stirring speed of 350 rpm. More importantly, the produced CO2 by waste crude glycerol fermentation was almost entirely captured in situ, which resulted in a 152-fold increase in the ratio of H2 to CO2. In this process, the conductivity of the fermentation broth was reduced significantly in Ca(OH)2 group because of the generation of insoluble CaCO3. When ammonium hydroxide was added in the 5 M Ca(OH)2 group as an inorganic nitrogen source instead of (NH4)2SO4, the conductivity of the fermentation broth was reduced by 54.3% when compared to the 5 M NaOH group. The soluble protein concentration of fermentation broth was obviously decreased by 44.1%, while 5 M Ca(OH)2 suspension was employed as the CO2 capture agent and pH regulator rather than 5 M NaOH. The reduced soluble salt and protein concentrations in the fermentation broth will significantly contribute to the downstream product separation process. Undoubtedly, the pressure of wastewater treatment would also be relieved, promoting the green production process. This new process integrates the generation of important chemicals , material (CaCO3) and clean energy (H2) using waste glycerol with a dramatically reduced carbon footprint. Therefore, this process provides a green and environmentally friendly production method for 1,3-PDO, which is highly consistent with sustainable development goals and serves as a valid reference for the production of bio-based chemicals to achieve net-zero CO2 emissions. It will broaden the range of applications for bio-based chemicals.Higher concentrations of 1,3-PDO (> 80.0 g/L) were accomplished from waste crude glycerol by C. butyricum DL07, previously selected from anaerobic active sludge [e sludge , was usee sludge , but amme sludge .C. butyricum DL07 was revived and cultured in seed medium. 4% (v/v) of strain suspension was inoculated into the seed medium. The seed culture was carried out in shaker at 37 °C and 200 rpm for 12 h. Fermentations were performed in a 5.0 L bioreactor  containing 2.0 L fermentation medium. To ensure an anaerobic environment, N2 was bubbled into the fermentation medium at 0.15 vvm, starting at 1 h before inoculation, and then stopped at 1 h after inoculation. 10% (v/v) of inoculum was inoculated into the bioreactor. The bioreactor was automatically run at various stirring speeds according to the experimental design. Throughout the fermentation process, the pH was maintained by different concentrations of Ca(OH)2 suspension involving NaOH and ammonia (6.80 g/L). Ca(OH)2 suspension was stirred at 100 rpm during service period.3 deposits were collected by centrifugation at 5000 rpm for 10 min.Fed-batch fermentations were carried out using continuous feeding strategy. The initial glycerol concentration was 40 g/L. When the residual glycerol concentration dropped to 20 g/L, crude glycerol was manually pumped into the bioreactor to maintain the glycerol concentration of about 20 g/L during the fermentation. The detailed operation of fermentation can refer to the previous report . The exh2SO4) and sample volume were 35 °C, 65 °C, 0.6 mL/min and 20 μL, respectively. Each testing sample from the fermentation broths was centrifuged for 10 min at 12,000 r/min. The clarified fermentation broth and chloroform were mixed at a ratio of 1:1 and centrifuged again under the above conditions to remove soluble proteins. After proper dilution, the above sample was filtered through a 0.22 μm membrane filter for analysis. All the precipitates were washed twice with pure water to remove impurities before being dried at 50 °C until constant weight for the collection of CaCO3. The collected exhaust gas was detected by gas chromatography . The conductivity of fermentation broth was measured using an intelligent conductivity meter. The ions in the fermentation broth were analyzed using high-performance ion chromatography. The concentration of soluble proteins contained in the fermentation broth was determined by the bradford protein assay kit .Cell mass was represented by the optical density of the sample at 650 nm using a UV–visible spectroscopy system . The concentrations of 1,3-PDO, glycerol and acids  were analyzed by high performance liquid chromatography  equipped with an Aminex HPX 87H column (300 × 7.8 mm) , a differential refractometer (Waters 2414) and an autosampler (Waters 2707). Operating conditions applied for detector temperatures, column temperature, the flow rate of mobile phase (5 mM H2 (mol) in the fermentation was calculated according to the following formula. Where consumption of NaOH is the consumption of NaOH in the fermentation. Total organic acids are the total organic acids produced in the fermentation.The fixed CO3 was examined by a scanning electron microscopy . The polymorphs of CaCO3 particles were characterized via X-ray diffraction (XRD) with Rigaku D/max 2400 V diffractometer (Japan).The elemental composition of precipitations was analyzed by energy dispersive spectrometer (EDS) equipped with an SEM instrument. The morphology of CaCOAdditional file 1: Fig. S1 Production of lactic acid using different CO2 capture agents. The solid symbol represents lactic acid production, and the hollow symbol represents citric acid consumption. The stirring speed is 250 rpm in every groups. Fig. S2 XRD pattern of CaCO3 from the fermentation. Fig. S3 Ratio of H2 and CO2 in exhaust gas using different CO2 capture strategies. Fig. S4 1,3-PDO production under different CO2 capture scenarios. NaOH represents 5 M NaOH solution; Ca(OH)2 represents 5 M Ca(OH)2 solution; In the ammonium hydroxide group, ammonium hydroxide acted as an inorganic nitrogen source and, otherwise, (NH4)2SO4 acted as an inorganic nitrogen source.