Rational highly dispersed ruthenium for reductive catalytic fractionation of lignocellulose

Producing monomeric phenols from lignin biopolymer depolymerization in a detachable and efficient manner comes under the spotlight on the fullest utilization of sustainable lignocellulosic biomass. Here, we report a low-loaded and highly dispersed Ru anchored on a chitosan-derived N-doped carbon catalyst (RuN/ZnO/C), which exhibits outstanding performance in the reductive catalytic fractionation of lignocellulose. Nearly theoretical maximum yields of phenolic monomers from lignin are achieved, corresponding to TON as 431 molphenols molRu−1, 20 times higher than that from commercial Ru/C catalyst; high selectivity toward propyl end-chained guaiacol and syringol allow them to be readily purified. The RCF leave high retention of (hemi)cellulose amenable to enzymatic hydrolysis due to the successful breakdown of biomass recalcitrance. The RuN/ZnO/C catalyst shows good stability in recycling experiments as well as after a harsh hydrothermal treatment, benefiting from the coordination of Ru species with N atoms. Characterizations of the RuN/ZnO/C imply a transformation from Ru single atoms to nanoclusters under current reaction conditions. Time-course experiment, as well as reactivity screening of a series of lignin model compounds, offer insight into the mechanism of current RCF over RuN/ZnO/C. This work opens a new opportunity for achieving the valuable aromatic products from lignin and promoting the industrial economic feasibility of lignocellulosic biomass.

As noted, the study is very well done and very extensive and would provide a very interesting addition to the literature on lignin first; impact and novelty wise a stronger case needs to be made in my opinion to be suitable for publication in Nature Commun.
Please find some additional comments below: -While not part of course of the assessment of the merits of the paper, the manuscript is in need of a very careful and extensive language check -The authors should more explicitly compare their results with (own) literature; this holds for the catalyst synthesis part, but also for the use of a Lewis acid cocatalyst (cf. the system by Abu-Omar which contains Pd+Zn) -It would be interesting to provide more information on the dimer/trimer/oligomer components of the lignin oil obtained; the group of Sels have recently reported extensive analyses that could of use here.
Reviewer #2 (Remarks to the Author): The authors present a new Ru catalyst for reductive catalytic fractionation of lignocellulosic materials. The results are fascinating and provide further insight into the relationships between catalyst and lignin's active stabilisation. The experimental evidence supports the claims. However, it is unclear how the authors could separate the catalyst from the pulp (on page 15, "To evaluate the reusability of RuN/ZnO/C, the spent catalyst isolated from the carbohydrate pulp was submitted for birch RCF directly"). The experimental details need to be presented in Experimental since catalyst recovery in RCF is still very problematic. In summary, it is an exciting manuscript with very nice results.
Reviewer #3 (Remarks to the Author): The authors present a nice study on developing a Ru single atom catalyst for reductive catalytic fractionation of biomass. The manuscript needs to be edited for minor grammatical mistakes (e.g., articles). The manuscript handles the synthesis of a new catalyst, detailed characterization of the catalyst, the activity of the catalyst in the hydrogenolysis of birch sawdust, effect of reaction conditions on hydrogenolysis yield, effect of solvent and comparison of different biomass sources. Authors put effort on completing the picture of several aspects of the new catalyst and compared it to standards catalysts. It is a very dense manuscript, however there are some points need to be clarified. The manuscript could be considered for Nature Communications after addressing the issues listed below: 1. For the determination of the biomass composition, NREL procedure was used. The total composition obtained was found to be 87.2%. This looks a bit low for total composition analysis. Generally, it would be 95+% by the NREL protocol. Why this is so low? Did you grind the biomass to the conditions? 2. Is the catalyst pre-reduced before the reaction? If not, the authors should comment on the catalyst oxidation state under reaction conditions. One would expect methanol reforms and reduces the catalyst; however, catalyst characterization does not clarify this. Authors should comment of this important aspect of the catalyst. Figure 4c: Liquid phase reactions are expected not to be affected by the pressure change. At the pressures studied, majority of the reaction mixture is in the gas phase. The authors should provide thermodynamic calculations for the conditions used. Also, are these the final pressures or the set pressures at the room temperature? 5-On page 22, the procedure for the time profile experiment is described. It is specified that the reaction pressure was kept at 3 MPa at 200 oC. According to the phase diagram, under these conditions, methanol is expected to be completely in gas phase. Is 3 MPa your operating pressure? If so, this complicates the reaction system, and may affect the reliability of the results.

Regarding
6-Supplementary Information, page 4 describes the analytical procedure for calculating the monomer yield. In the first paragraph, instead of "…available standards or independent synthesis.", "…available standards or independently synthesized standards." would be easier to understand. 7-In the formula given in supplementary information, on page 4, what does M (total monomers) correspond to? Is this the mass of the detected and quantified monomers (e.g. Pr-G, Pe-G, POH-S,…) or is it referring to the mass after converting the moles of quantified monomers to the moles of monolignols (coniferyl alcohol, sinapyl alcohol) and their corresponding mass? The equation should be referring to the mass of the monolignols found in the biomass to determine the weight percent of Klason lignin. The authors report on the use of a single atom/atomically dispersed Ru catalyst for the ligninfirst reductive catalytic fractionation of various kinds of woody biomass feedstock, with particular emphasis on birch. Close to theoretical monomer yields are obtained, with very high selectivities to the propylguaiacol and syringol monomers. Overall, the study is very comprehensive and well executed. The catalyst is extensively characterized, including the necessary HAAFD-STEM and XAS analyses, and the lignin-first studies are extensive, detailing full biomass utilization, feedstock variation, catalyst reuse and some model studies.
The catalyst is synthesized from a chitosan C-source with Zn(OAc)2 added to give the additional Lewis acidity, a feature of the catalyst that later proves quite essential. It should be noted that the authors previously (in Nat Commun, ref 42) reported on a MOF-derived atomically dispersed Ru/ZnO/C catalyst and used this system in C-lignin hydrogenolysis. In the previous contribution, catalyst stability and reuse was not covered. In the present study, the remaining N-functional groups in the carbon support are suggested to provide additional stability to the single Ru sites, which would then be the novel aspect of this particular catalyst system.
Overall, the high, close to theoretical yields have been obtained before in reported RCF efforts with traditional nanoparticulate carbon-supported precious metal catalysts. The impact of the paper then hinges on the combination of efficient precious metal use and the intrinsic stability of the catalyst, i.e. the ability for extensive reuse. The authors report on a regeneration method, which restores activity to close the original values, at least for one run, after this a small gradual drop in activity is seen in Fig 6. Emphasis is put on the stability of the Ru single sites, but the elemental analysis reported in the SI do show considerable leaching of the Zn Lewis acid component, likely simply due to leaching. This is not commented on much in the main text. I am therefore hesitant as to if the main advance, i.e. the development of a stable atomically dispersed catalyst for RCF, is sufficiently demonstrated by the results. This would require a comparison with the MOF derived catalyst, a more extensive study on the fate and influence of the (soluble?) Zn component and more extensive reuse.

Reply:
The reviewers' point, that is the leaching of the Zn component, is right. This was confirmed by the ICP-analysis of RCF reaction solution with fresh RuN/ZnO/C, in which Zn element was detected. Of note, a slowdown of Zn leaching was observed in further cycles, as seen in the cases of fresh, 1 st recycled, and 2 nd recycled (after calcinated regeneration) catalysts, the Zn contents were determined as 6.2, 4.9, and 4.5 wt%, respectively. In our previous work, we have reported that the calcination of spent catalyst can suppress further leaching of Zn during lignin hydrogenolysis (ChemSusChem, 2018(ChemSusChem, , 11, 2114. Catalytic results indicated that the partial loss of Zn did not influence catalytic performance dramatically in birch RCF. The soluble Zn might not promote the lignin hydrogenolysis, because the combination of RuN/C and Zn(OAc)2 did not show higher performance than RuN/C in terms of activity and selectivity.
As suggested, we used MOF-derived Ru catalyst (Ru/ZnO/C-MOF) to treat birch under RCF conditions, which gave ca. 35 wt% yield of phenolic monomers with propenyl side-chain as major products. The hydrothermally treated Ru/ZnO/C-MOF catalyst showed a slight drop in monomers yields (ca. 30%). By comparison, H.T.-RuN/ZnO/C (reported in this manuscript) showed a consistent performance with fresh one. In this context, the coordinated N species in RuN/ZnO/C can enhance the stability of Ru centers.
These results have been added in the manuscript. We hope our responses will satisfy you.
As noted, the study is very well done and very extensive and would provide a very interesting addition to the literature on lignin first; impact and novelty wise a stronger case needs to be made in my opinion to be suitable for publication in Nature Commun.
Please find some additional comments below: -While not part of course of the assessment of the merits of the paper, the manuscript is in need of a very careful and extensive language check.
Reply: Thanks for pointing out these problems. We have carefully checked the manuscript and English language has been edited by Author Services of Springer Nature.
-The authors should more explicitly compare their results with (own) literature; this holds for the catalyst synthesis part, but also for the use of a Lewis acid cocatalyst (cf. the system by Abu-Omar which contains Pd+Zn).
Reply: As suggested, we tested the catalytic performance of the combination of RuN/C and Zn(OAc)2 (Abu-Omar's system) in RCF of birch, by which phenolic monomers were obtained in 21.4 wt% yield. This result is similar to that from RuN/C (24.9 wt%), and lower than that from RuN/ZnO/C (46.4 wt%), suggesting that the anchored ZnO species serve an auxiliary role in lignin hydrogenolysis. These results and discussions have added in the manuscript and Supplementary   Table 4. -It would be interesting to provide more information on the dimer/trimer/oligomer components of the lignin oil obtained; the group of Sels have recently reported extensive analyses that could of use here.

Reply:
In GPC profiles, two major signals ca. 183 and 428 g mol -1 were ascribed to phenolic monomers and dimers, and a small broad peak corresponding to oligomers was also observed.
To analyze the dimers, the lignin oils were silylated and characterized on GC-MS. Supported by literatures, a total of 11 different dimers were identified, among which most had C-C linkages, except for a small number of 4-O-5 dimer.
These results have been added in the manuscript and Supplementary Information. Thank you for your professional suggestion.
Reviewer #2 (Remarks to the Author): The authors present a new Ru catalyst for reductive catalytic fractionation of lignocellulosic materials. The results are fascinating and provide further insight into the relationships between catalyst and lignin's active stabilisation. The experimental evidence supports the claims. However, it is unclear how the authors could separate the catalyst from the pulp (on page 15, "To evaluate the reusability of RuN/ZnO/C, the spent catalyst isolated from the carbohydrate pulp was submitted for birch RCF directly"). The experimental details need to be presented in Experimental since catalyst recovery in RCF is still very problematic.
In summary, it is an exciting manuscript with very nice results.

Reply: Thanks very much for this important suggestion.
After RCF reaction, the solid phase containing carbohydrate pulp and catalyst was dried at room temperature, which was then transferred to a 100-mesh screening. The spent catalyst could be readily separated from the carbohydrate pulp by sieving, because the carbohydrate remained the original framework of biomass (2-5 mm) without collapse. The detailed scheme and procedure for the separation of catalyst and pulp have been added as Supplementary Fig. 25 (as shown below).

Reviewer #3 (Remarks to the Author):
The authors present a nice study on developing a Ru single atom catalyst for reductive catalytic fractionation of biomass. The manuscript needs to be edited for minor grammatical mistakes (e.g., articles). The manuscript handles the synthesis of a new catalyst, detailed characterization of the catalyst, the activity of the catalyst in the hydrogenolysis of birch sawdust, effect of reaction conditions on hydrogenolysis yield, effect of solvent and comparison of different biomass sources.
Authors put effort on completing the picture of several aspects of the new catalyst and compared it to standards catalysts. It is a very dense manuscript, however there are some points need to be clarified. The manuscript could be considered for Nature Communications after addressing the issues listed below: 1. For the determination of the biomass composition, NREL procedure was used. The total composition obtained was found to be 87.2%. This looks a bit low for total composition analysis.
Generally, it would be 95+% by the NREL protocol. Why this is so low? Did you grind the biomass to the conditions?
Reply: Thank you for your kind comment. The biomass sawdust has been ground and screened through 60-mush sifter before NREL determination. If extracted components (3.5 wt%) was added, the total composition of birch would reach 90.7 wt%, albeit without considering water and ash. After comparison, current total composition obtained from birch is comparable to those reported in literatures (as shown below).
2. Is the catalyst pre-reduced before the reaction? If not, the authors should comment on the catalyst oxidation state under reaction conditions. One would expect methanol reforms and reduces the catalyst; however, catalyst characterization does not clarify this. Authors should comment of this important aspect of the catalyst.
Reply: please see the following response. 3. Related with the question 2, do you have any EXAFS study related with the reduction of Ru atoms? Did you perform the experiments before and after reduction and see the differences?

Reply to questions 2 and 3:
(1) In the manuscript, the RuN/ZnO/C catalyst was used directly without pre-reduction. As suggested, we treated RuN/ZnO/C in MeOH at 240 °C , H2 (3 MPa at R.T.) for 4 h, which gave RuN/ZnO/C-(R) catalyst. In RCF of birch, RuN/ZnO/C-(R) can produce phenolic monomers in 46.1 wt% yield with 83% selectivity to Pr-G and Pr-S, being in line with RuN/ZnO/C.
(2) To realize the oxidation state of Ru in RCF reactions, we performed XAFS measurements for RuN/ZnO/C-(R) (see below). The Ru K-edge XANES spectra showed that the near-edge of RuN/ZnO/C-(R) was located between Ru foil and RuO2, revealing the positively charged state of Ru. The EXAFS spectrum of the Ru K-edge in R space displayed a primary peak situated at 2.3 Å, corresponding to Ru−Ru coordination. These results were in accord with the scenarios in HAADF-STEM images, where both atomically dispersed Ru single atoms and Ru nanoclusters were detected ( Supplementary Fig. 5). Obviously, most of the single Ru atoms were reduced into nanoclusters during pre-reduction process.
(3) Since there was no difference of catalytic performance between RuN/ZnO/C and RuN/ZnO/C-(R), Ru nanoclusters in RuN/ZnO/C-(R) should be the active species for lignin hydrogenolysis. The as-formed Ru nanoclusters should be beneficial to the dissociation of H2, an uphill process in lignin hydrogenolysis, because H2 dissociation on nanoparticles via homolytic path is more convenient than that on single atoms via heterolytic path.
Given XAFS measurements of RuN/ZnO/C and RuN/ZnO/C-(R) were respectively performed at Shanghai Synchrotron Radiation Facility (SSRF) and Beijing Synchrotron Radiation Facility (BSRF) (SSRF was closed recently due to COVID-19), we did not combine the XANES spectra.
We really appreciate this reviewer for this forward-looking question, which offered insight into understanding the possible pathway of lignin hydrogenolysis under such a catalyst. These results and discussion have been added in the manuscript.  Supplementary Table 8).
We calculated the mole fraction solubility of H2 in MeOH based on a reported model, which displayed a linear relationship with the total monomers yields from RCF of birch ( Supplementary   Fig. 17, see also blow). This indicated that sufficient H2 transfer from gas to liquid phase is necessary for current lignin hydrogenolysis.
We are grateful for this reviewer for pointing out these issues. These results and discussions have been added in the manuscript and Supplementary Information. If so, this complicates the reaction system, and may affect the reliability of the results.
Reply: Thanks for pointing out this mistake. 3 MPa is the set H2 pressure at the room temperature, and the final pressure was kept at 7.2 MPa (this reaction was performed in a 300 mL reactor with a constant pressure complementary H2 device). Phase diagram suggested that MeOH kept as a liquid phase under such a condition. This mistake has been revised in the manuscript.
6. Supplementary Information, page 4 describes the analytical procedure for calculating the monomer yield. In the first paragraph, instead of "…available standards or independent synthesis.", "…available standards or independently synthesized standards." would be easier to understand.

Reply:
The description of "…available standards or independent synthesis." has been revised as "…available standards or independently synthesized standards.". before GC and GC-MS systems. Linear calibration curves were produced from 0.14 mg/mL to 3.9 mg/mL for authentic samples. Reply: The standard curve was made by polystyrene standards having different molecular weights, including 162 g mol -1 , 370 g mol -1 , 580 g mol -1 , 860 g mol -1 , and 1320 g mol -1 . The polystyrene standards were purchased from Agilent Technologies (polystyrene calibration kit S-L2-10, Part

In the formula given in supplementary information
Number PL 2010-0105). The detailed information on polystyrene standards has been described in the Supplementary Information.

10
. Table 1 of supplementary information lists the ICP-AES results of the fresh and spent catalyst.
It looks like some of the Zn leaches. Was that confirmed by analyzing the liquid mixture? The authors should comment on the effect of Zn leaching on catalyst stability.
Reply: Thanks for pointing out this. As suggested, we measured the RCF reaction solution (obtained from fresh RuN/ZnO/C) by ICP-analysis, in which Zn element was detected. This result confirmed that some of the Zn has leached. We also noticed that Zn leaching become slow in further cycles, as seen in the cases of fresh, 2 nd use, and 3 rd use (after calcinated regeneration) catalysts, the Zn contents were determined as 6.2, 4.9, and 4.5 wt%, respectively. Catalytic results