Autoxidation Catalysis for Carbon–Carbon Bond Cleavage in Lignin

Selective lignin depolymerization is a key step in lignin valorization to value-added products, and there are multiple catalytic methods to cleave labile aryl–ether bonds in lignin. However, the overall aromatic monomer yield is inherently limited by refractory carbon–carbon linkages, which are abundant in lignin and remain intact during most selective lignin deconstruction processes. In this work, we demonstrate that a Co/Mn/Br-based catalytic autoxidation method promotes carbon–carbon bond cleavage in acetylated lignin oligomers produced from reductive catalytic fractionation. The oxidation products include acetyl vanillic acid and acetyl vanillin, which are ideal substrates for bioconversion. Using an engineered strain of Pseudomonas putida, we demonstrate the conversion of these aromatic monomers to cis,cis-muconic acid. Overall, this study demonstrates that autoxidation enables higher yields of bioavailable aromatic monomers, exceeding the limits set by ether-bond cleavage alone.

In summary, this manuscript contributes new knowledge to the field of lignin degrada�on while simultaneously expanding the footprint of the reduc�ve cataly�c frac�ona�on process and u�liza�on of raw material lignin.It is a well-inves�gated project.The manuscript is presented with solid data and communicated clearly.I have some sugges�on below and hope they will make the manuscript beter.

1)
The lignin field has alterna�ve oxida�on strategies.For example, Stahl has showed that TEMPOmediated beta-O-4 oxida�on.What is the comparison between these two methods?What are the benefits of the cataly�c autoxida�on approach?

2)
Those reac�on schemes have the same condi�ons, I recommend redraw these figures, and make a tabulated data including several columns: substrate, condi�ons, products, and results.These will make them much nicer.

Comments to the Author
The manuscript by Gu and coworkers describes the conversion of lignin to muconic acid through a series of reac�ons including frac�ona�on to produce lignin oligomers (+monomers), acetyla�on to form acetylated oligomers (+monomers), removal of monomers to avoid monomer degrada�on, the cataly�c autoxida�on (central focus) and bioconversion of products to muconic acid (secondary focus).While the condi�ons used here have been used in previous studies, the demonstra�on of oxida�ve degrada�on on lignin oligomers makes this approach more advantageous and successful.The authors examine some model studies to predict the product outcomes from the RCF oils, some degrada�on studies to determine the degrada�on pathways of the products (ini�ally the product yields are lower than the star�ng content of the RCF oils).Ul�mately, this results in the authors separa�ng out the monomers from the RCF oil to study the degrada�on of the oligomers/dimers.A single catalyst system with some inves�ga�on into catalyst loading/temperature provides op�mal yield resul�ng in a 17% increase of monomers.The authors then subject the oxida�on products to Pseudomonas pu�da cultures to produce muconic acid.The work is thorough and rigorous, but more experiments are needed to bring this to the level expected for ACS Central Science; as is, this report would be more suitable for ACS Sustainable Chemistry & Engineering.
-Did the authors subject the RCF oils to Pseudomonas pu�da cultures in the absence of the acetyla�on/oxida�on protocol?Given the modest yields from oxida�on, perhaps direct bioconversion could produce similar or higher yields of muconic acid.
-There is limited mechanis�c analysis, ra�onaliza�on for product outcomes (i.e. 5, what is the mass balance, di-carboxylic acids, no ac�va�on at the bridge posi�on?) -Did the authors try NaCl, rather than NaBr, or something else with a larger BDE?A beter C-H abstractor might facilitate conversion of oligomers to products.Unless the authors hypothesize that oxygencentered radicals are primarily involved in the C-H abstrac�on step.
-Can the authors hypothesize a mechanism for acetate deprotec�on?The dis�lla�on step to separate out monomers is undesirable and a major limita�on, as the authors state in their conclusion; could the authors use a mixture of ace�c acid/ace�c anhydride as the solvent?
-Schemes 1-3 are redundant with regards to "same condi�ons" listed above everything but the first arrow.Perhaps there is a cleaner way to present these, with a generic scheme and reactants/products listed underneath.

Comments to the Author
The authors present a great study showing how a a side-stream from lignin depolymeriza�on in the "lignin-first" approach, that is RCF, can be converted to a mixture that can be addressed by biological funneling.Here, this entails an oligomeric frac�on obtained a�er acetyla�on and removal of valuable monomers by dis�lla�on, which is subsequently oxidized using Co/Mn/Br-based cataly�c autooxida�on.The product mixture from the auto oxida�on could be converted to cis,cis-muconic acid using P. pu�da previously studied by some of the authors involved.Overall this study displays an innova�ve approach to u�lize this otherwise low-value side stream obtained from a cataly�c lignin conversion process that has the poten�al to be scaled in the near future.There can be some concerns raised over the overall efficiency and the "greeness".But as a proof of concept this paper is the first of its kind establishing the possibility to link this bio-based chemical side stream to biological funneling.This can be a great star�ng point for the development of new innova�ve approaches for the infidividual steps to reach an overall sustainable and green process for conver�ng lignocellulosic streams in emerging biorefining schemes.These aspects combining different fields of cataly�c conversion and biological funneling make this manuscript highly suitable for publica�on in ACS Central Science.

Author's Response to Peer Review Comments:
Reviewer comments are provided in black font.Our responses are provided in blue font.

Reviewer 1
Beckham, Stahl and co-workers have prepared a manuscript �tled 'Autoxida�on catalysis for carboncarbon bond cleavage in lignin' describing a novel approach of u�lizing Co/Mn/Br-based catalyst to promote autoxida�on of lignin.The state-of-the-art approach, reduc�ve cataly�c frac�ona�on, targets C-O bond cleavage, while the authors showed their methods can facilitate C-C bond cleavage instead, therefore giving higher yields of aroma�cs.Instead of designing new catalysts, the authors applied an exis�ng oxida�on cataly�c system in industry (para-xylene to terephthalic acid process) to an unsolved problem, lignin depolymeriza�on, and they showed promising results.
In summary, this manuscript contributes new knowledge to the field of lignin degrada�on while simultaneously expanding the footprint of the reduc�ve cataly�c frac�ona�on process and u�liza�on of raw material lignin.It is a well-inves�gated project.The manuscript is presented with solid data and communicated clearly.I have some sugges�on below and hope they will make the manuscript beter.
We thank the reviewer for the positive response to our work and for the constructive comments, which we have addressed below and through edits to the manuscript.

1)
The lignin field has alterna�ve oxida�on strategies.For example, Stahl has showed that TEMPOmediated beta-O-4 oxida�on.What is the comparison between these two methods?What are the benefits of the cataly�c autoxida�on approach?
The referenced study from Stahl et al. focused on ether bond cleavage, and we do not anticipate that this chemistry (TEMPO oxidation followed by formic acid-catalyzed ether bond cleavage) would be able to cleave C-C bonds, which was the focus of the current study.
More generally, a compara�ve study with other lignin oxida�on strategies is outside the scope of the present study, but we agree that such studies would be valuable and will be pursued in future work.We chose to study catalytic autoxidation in this work given its industrial relevance.The catalytic conditions used in this study (with acetic acid, 2 h residence time, and 120 °C with Co/Mn/Br) is directly inspired by the Amoco process conditions for the manufacture of terephthalic acid from p-xylene at ~80 MMT per year scale, here conducted with a lower temperature.By demonstrating that lignin oxidation can indeed be achieved with similar conditions, we aim to further develop this process toward industrial viability.
Previous research on Amoco oxidation conditions indicate that acetic acid can undergo degradation to CO or CO2, but the losses are minimal even when run up to 175-225 °C, which, as we show in our work, is higher than the temperature needed for C-C bond cleavage in lignin (Sheehan, R.J. Ullmann's Encyclopedia of Industrial Chemistry, 2012).It is also known that the Co/Mn/Br oxidation catalyst can be re-used for many years with little makeup (Tomás, R. A. F. et al. Chem. Rev. 2013, 113, 7421-7469).We are optimistic that the same beneficial features will be true for the present system.Work is ongoing to further optimize this process for lignin now, including transitioning to a flow-based process.

2)
Those reac�on schemes have the same condi�ons, I recommend redraw these figures, and make a tabulated data including several columns: substrate, condi�ons, products, and results.These will make them much nicer.
We thank the reviewer for this suggestion and have updated Schemes 1-3 to a table format, as shown in the uploaded copy with red highlights.

Reviewer 2
The manuscript by Gu and coworkers describes the conversion of lignin to muconic acid through a series of reac�ons including frac�ona�on to produce lignin oligomers (+monomers), acetyla�on to form acetylated oligomers (+monomers), removal of monomers to avoid monomer degrada�on, the cataly�c autoxida�on (central focus) and bioconversion of products to muconic acid (secondary focus).While the condi�ons used here have been used in previous studies, the demonstra�on of oxida�ve degrada�on on lignin oligomers makes this approach more advantageous and successful.The authors examine some model studies to predict the product outcomes from the RCF oils, some degrada�on studies to determine the degrada�on pathways of the products (ini�ally the product yields are lower than the star�ng content of the RCF oils).Ul�mately, this results in the authors separa�ng out the monomers from the RCF oil to study the degrada�on of the oligomers/dimers.A single catalyst system with some inves�ga�on into catalyst loading/temperature provides op�mal yield resul�ng in a 17% increase of monomers.The authors then subject the oxida�on products to Pseudomonas pu�da cultures to produce muconic acid.The work is thorough and rigorous, but more experiments are needed to bring this to the level expected for ACS Central Science; as is, this report would be more suitable for ACS Sustainable Chemistry & Engineering.
We thank the reviewer for the positive response to our work and for the constructive comments, which we have addressed below and through edits to the manuscript.
-Did the authors subject the RCF oils to Pseudomonas pu�da cultures in the absence of the acetyla�on/oxida�on protocol?Given the modest yields from oxida�on, perhaps direct bioconversion could produce similar or higher yields of muconic acid.
We appreciate this idea from the reviewer, given that monomers are indeed present alongside the dimers and oligomers found in RCF oil.As we described in a perspec�ve from 2016, lignin oxida�on lends itself to biological funneling much more so than lignin-derived substrates from reduc�ve chemistry (Beckham, G.T. et al., Current Opin. Biotechnol. 2016, 42, 40-53).
For RCF oil specifically, the use of this substrate for P. putida presents two major challenges: First, our group and others have shown that RCF oil is highly toxic to P. putida and other bacteria, likely due to the presence of toxic solvents and aroma�cs derived from reduced lignin (Wu, Y. et al., Chem Eng J, 2023, 452, 139267).In fact, RCF oil has been proposed as an industrial an�microbial due to its strong bacteriosta�c effect against pathogens like Staphylococcus aureus, even at concentra�ons as low as 0.25 wt% (Ebikade, E.O. et al., Green Chem, 2020, 22, 7435-7447).
Addi�onally, the oil itself is poorly soluble in water, making it difficult to incorporate into the aqueous media used for bacterial cul�va�on.In studies where RCF oil has been applied as a growth substrate for bacteria, it was solubilized in DMSO (which is also toxic to P. putida) and could only be added to aqueous media at a low concentra�on of 250 ppm (Fetherolf, M.M. et al., Proc Natl Acad Sci USA, 2020, 117, 25771-25778).
-There is limited mechanis�c analysis, ra�onaliza�on for product outcomes (i.e. 5, what is the mass balance, dicarboxylic acids, no ac�va�on at the bridge posi�on?) We agree that mechanistic studies and further investigation into product profiles could play an important role in future optimization of this process and will be pursued in future work.Importantly, given the similarity between the conditions used here and the commercial Amoco process, we expect the central mechanistic features to remain the same between the two systems (see refs. 26 and 27).
-Did the authors try NaCl, rather than NaBr, or something else with a larger BDE?A beter C-H abstractor might facilitate conversion of oligomers to products.Unless the authors hypothesize that oxygencentered radicals are primarily involved in the C-H abstrac�on step.
We selected the Co/Mn/Br catalyst for this work given its relevance to industrial autoxidation processes, as noted above.The C-H abstraction step under such autoxidation conditions is believed to involve both Br-and oxygencentered radicals (Tomás, R. A. F. et al. Chem. Rev. 2013, 113, 7421-7469).The assessment of different radical mediators, such as NaCl, is outside the scope of the present study, but we agree with the reviewer that such studies could be valuable and will be pursued in future work.
-Can the authors hypothesize a mechanism for acetate deprotec�on?The dis�lla�on step to separate out monomers is undesirable and a major limita�on, as the authors state in their conclusion; could the authors use a mixture of ace�c acid/ace�c anhydride as the solvent?
Under Amoco autoxidation conditions, O2 is the terminal oxidant and generates water as a byproduct.Hydrolysis of the acetyl groups is a likely pathway for deprotection, and we have updated the discussion on pg. 3 of the main text to note this: "Deprotection of the acetyl groups likely occurs through hydrolysis, as water is a byproduct under AMOCO oxidation conditions. 22" We are interested in exploring the use of ace�c acid/ace�c anhydride solvent for the in situ protec�on of phenol groups, which will be pursued in future work.
-Schemes 1-3 are redundant with regards to "same condi�ons" listed above everything but the first arrow.Perhaps there is a cleaner way to present these, with a generic scheme and reactants/products listed underneath.We thank the reviewer for this suggestion and have updated Schemes 1-3 to table format.