Effects of Lignocellulosic Biomass-Derived Hydrolysate Inhibitors on Cell Growth and Lipid Production During Microbial Fermentation of Oleaginous Microorganisms—A Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review "Effects of lignocellulosic biomass-derived hydrolysates inhibitors on cell growth and lipid production during microbial fermentation of oleaginous microorganisms – A review" is devoted to the current topic of the conversion of lignocellulosic raw materials to lipid production and the role of inhibitors in this process that can be formed during the preliminary treatment of this raw material. The authors analyzed 134 references, of which 42 (31% in total) were published in 2020-2024. The review consists of separate sections, which, in the authors' opinion, will allow readers to quite easily understand a complex topic and develop their own technical solutions when considering a specific source of lignocellulose as a raw material. During the review process, I discovered that this topic has already been published as a chapter in a book (DOI: 10.1007/978-981-96-0982-6_7). I do not consider this fact critical. But for the publication of this review, I have recommendations for correcting the submitted manuscript. The list is given below.

1. It is recommended to revise the list of publications of this review and increase the number of fresh references with the release of 2020-2025, possibly by excluding "outdated" sources with all due respect to the authors of these sources.
2. It is recommended to exclude from the text of the review the description of the conversion of non-lignocellulosic sources, such as "starch" or "potatoes". The authors do not disclose the problems of enzymatic hydrolysis of pre-treated lignocellulose. These problems do not concern the enzymatic hydrolysis of starch.
3. It is recommended to choose a place in the review (preferably in the introduction) and briefly describe the sequential pathway of lignocellulose conversion into lipid production, in which to indicate the location of the inhibitors: in the solid phase or in the liquid phase. It will be unclear to the reader how inhibitors, which should be in the liquid phase after preliminary processing of the raw material, will affect the enzymatic hydrolysis of the solid residue, and then the fermentation of the nutrient medium in lipid production.
4. Given the outlook of the readers of the publication, Figure 1 should be replaced with a process diagram indicating the stage and state of the phase (liquid or solid) in which the inhibitors are formed.
5. The review does not provide examples of calculating the yield of lipids based on the source of lignocellulose depending on the inhibitor.
6. When describing the methods of preliminary processing, the main statement is missing: what types of processing lead to the production of substances that reduce the efficiency of the process (true inhibitors).
7. It is recommended to check the contents of Table 2: there are no data in the column "Lipid concentration de-creased (%)".
8. The review does not provide a typical idea of ​​the material balance of the process of converting lignocellulose into lipids in comparison with the theoretical process in the absence of inhibitors. Therefore, the Conclusions are descriptive and lack the technical recommendations that the reader needs. This situation needs to be corrected.
9. The review is very repetitive. Their exclusion would benefit the manuscript. Apparently, the review was written by several authors in separate sections. The text needs to be proofread and this situation corrected.

Author Response

Point 1: It is recommended to revise the list of publications of this review and increase the number of fresh references with the release of 2020-2025, possibly by excluding "outdated" sources with all due respect to the authors of these sources.

Response 1: Thank you for your valuable suggestion. Based on your comment, we have updated the reference list by incorporating more recent publications from 2020-2025 and replacing some outdated sources. Please check the revised manuscript for details.

• Page 2, Line 50-54: Recent study data has been provided “Recent studies have shown that oleaginous yeasts, such as Yarrowia lipolytica, can efficiently utilize lignocellulosic biomass hydrolysates for lipid production, achieving intracellular lipid contents of up to 42% (w/w) in bioreactor cultures. This demonstrates their potential as promising candidates for sustainable microbial lipid production [3].”

Added reference: Dias, B.; Fernandes, H.; Lopes, M.; Belo, I. Yarrowia lipolytica produces lipid-rich biomass in medium mimicking lignocellulosic biomass hydrolysate. Applied Microbiology and Biotechnology 2023, 107 (12), 3925-3937. https://doi.org/10.1007/s00253-023-12565-6

• Page 9, Line 351-357: “Additionally, theoretical studies indicate that the maximum lipid yield from glucose metabolism can reach 32%, assuming all acetyl-CoA is directed toward lipid biosynthesis [65,66]. However, under practical fermentation conditions, metabolic losses, inhibitory effects, and process inefficiencies reduce actual lipid yields to approximately 22% [67]. In lignocellulose-derived hydrolysates, the presence of additional inhibitors further reduces conversion efficiency, leading to yields significantly below theoretical expectations [68].”

Added reference: Ratledge, C. Biochemistry, stoichiometry, substrates and economics. Single cell oil 1988, 1.

Fakas, S.; Čertik, M.; Papanikolaou, S.; Aggelis, G.; Komaitis, M.; Galiotou-Panayotou, M. γ-Linolenic acid production by Cunninghamella echinulata growing on complex organic nitrogen sources. Bioresource Technology 2008, 99 (13), 5986-5990. https://doi.org/10.1016/j.biortech.2007.10.016

Papanikolaou, S.; Aggelis, G. Biotechnological valorization of biodiesel derived glycerol waste through production of single cell oil and citric acid by Yarrowia lipolytica. Lipid technology 2009, 21 (4), 83-87. https://doi.org/10.1002/lite.200900017

Daskalaki, A.; Perdikouli, N.; Aggeli, D.; Aggelis, G. Laboratory evolution strategies for improving lipid accumulation in Yarrowia lipolytica. Applied microbiology and biotechnology 2019, 103, 8585-8596. https://doi.org/10.1007/s00253-019-10088-7

• Page 16, Line 630-634: “Fed-batch bioreactors enable controlled substrate feeding, reducing inhibitor toxicity and improving lipid yields [133]. Furthermore, membrane bioreactors incorporating Sim-ultaneous Saccharification, Filtration, and Fermentation (SSFF) have demonstrated po-tential in maintaining high cell density while mitigating inhibitory effects, making them promising candidates for large-scale applications [133].”

Added reference: Li, C.; Chen, K.; Wang, B.; Nges, I. A. Bioreactor design for efficient biofuels production from lignocellulosic biomass. In Biofuels Production from Lignocellulosic Materials, Elsevier, 2025; pp 181-217. https://doi.org/10.1016/B978-0-443-16052-3.00013-1

 

Point 2: It is recommended to exclude from the text of the review the description of the conversion of non-lignocellulosic sources, such as "starch" or "potatoes". The authors do not disclose the problems of enzymatic hydrolysis of pre-treated lignocellulose. These problems do not concern the enzymatic hydrolysis of starch.

Response 2: Thank you for pointing this out. We have removed the descriptions related to non-lignocellulosic sources such as starch and potatoes to maintain the focus on lignocellulosic biomass conversion:

Page 2, Line 47-48: The sentence “…that cassava starch can undergo…” has been removed.

Page 4, Line 191-192: The sentence “This technique is applicable to biomass like sugarcane, sweet potatoes, bulrush residues, cotton stalks, and corn stover.” has been revised: “This technique is applicable to biomass like sugarcane, bulrush residues, cotton stalks, and corn stover.”

 

Point 3: It is recommended to choose a place in the review (preferably in the introduction) and briefly describe the sequential pathway of lignocellulose conversion into lipid production, in which to indicate the location of the inhibitors: in the solid phase or in the liquid phase. It will be unclear to the reader how inhibitors, which should be in the liquid phase after preliminary processing of the raw material, will affect the enzymatic hydrolysis of the solid residue, and then the fermentation of the nutrient medium in lipid production.

Response 3: We appreciate this insightful suggestion. In the revised manuscript, we have added a brief description in the introduction to outline the sequential pathway of lignocellulose conversion into lipid production, highlighting the locations of inhibitors in either the solid or liquid phase.

Page 2-3, Line 93-100: The paragraph “The conversion of lignocellulosic biomass into microbial lipids involves pretreatment, enzymatic hydrolysis, fermentation, and lipid extraction. During pretreatment, the breakdown of hemicellulose and lignin generates liquid-phase inhibitors such as furfural, hydroxymethylfurfural (HMF), and weak acids. While most inhibitors remain in the liquid phase, solid-phase lignin residues can also interfere with enzymatic hydrolysis, reducing sugar release. In the fermentation stage, residual inhibitors in the hydrolysate can hinder microbial growth and lipid accumulation. A clear understanding of how these inhibitors form and impact different stages is essential for optimizing lignocellulose-based lipid production.” was added in introduction.

 

Point 4: Given the outlook of the readers of the publication, Figure 1 should be replaced with a process diagram indicating the stage and state of the phase (liquid or solid) in which the inhibitors are formed.

Response 4: Thank you for the suggestion. We have revised Figure 1 to a process diagram that clearly indicates each stage and the corresponding phase (solid or liquid) where inhibitors are generated.

 

Point 5: The review does not provide examples of calculating the yield of lipids based on the source of lignocellulose depending on the inhibitor.

Response 5: Thank you for your comment. We have specific examples and data on how different inhibitors affect lipid yield.

In Effects of inhibitors on cell growth and microbial oil production, we have included:

• Page 8, Line 336-339: “For Schizochytrium HX-308, cell growth and lipid accumulation remain largely unaffected when furfural concentration is below 1.2 g/L. However, as the furfural concentration increases to 1.8 g/L, the cell dry weight (CDW) and total lipids (TL) decrease by 57.7% and 58.5%, respectively.”
• Page 10, Line 395-396: “Moreover, Cryptococcus curvatus can efficiently produce lipids from corn straw hydrolysate that contains 15.9 g/L acetate.”
• Page 10, Line 399-401: “When the concentrations of acetic acid and formic acid were set at 50.0 mM and 65.2 mM, Mortierella isabellina achieved maximum lipid concentrations of 10.13 g/L and 9.11 g/L, respectively.”

 

Point 6: When describing the methods of preliminary processing, the main statement is missing: what types of processing lead to the production of substances that reduce the efficiency of the process (true inhibitors).

Response 6: We appreciate this valuable feedback. We have supplemented our discussion on how different pretreatment methods contribute to the formation of inhibitors that negatively affect process efficiency.

Page 4, Line 150-166: “The choice of pretreatment method significantly affects the type and concentration of inhibitors generated. Dilute acid hydrolysis, commonly used to break down hemicellulose and improve sugar release, often leads to the formation of furfural and hydroxymethylfurfural (HMF) due to the degradation of pentose and hexose sugars, respectively. These compounds can inhibit microbial growth by interfering with enzymatic activity and metabolic pathways. Alkaline pretreatment, which primarily targets lignin removal, results in the release of phenolic compounds. These phenolics, originating from lignin degradation, are known to disrupt microbial cell membranes and inhibit enzyme function, thereby reducing lipid production efficiency. Steam explosion and ammonia fiber expansion, while effective in disrupting biomass structure, can also generate weak acids (e.g., acetic acid), phenolics, and furan derivatives, all of which pose additional stress on microbial metabolism. In contrast, ionic liquid pretreatment has gained attention as a potentially milder alternative, but residual ionic liquids may still be toxic to microorganisms, depending on their chemical composition and concentration. Under-standing how different pretreatment strategies contribute to inhibitor formation is essential for optimizing lignocellulosic biomass utilization and improving microbial lipid yields.”

 

Point 7: It is recommended to check the contents of Table 2: there are no data in the column "Lipid concentration decreased (%)".

Response 7: Thank you for your comment. We have carefully checked Table 2. We found that, there are some data in the column "Lipid concentration decreased (%), meanwhile some data are not provided by the references. So, we use “-” in the Table, please check the Table below:

Fermentation process	Microbial	Inhibitor	Inhibitor concentration	Biomass decline (%)	Lipid concentration decreased (%)	References
Microbial growth	Yarrowia lipolytica	acetic acid	75 mM	100	-	[103]
	Yarrowia lipolytica	formic acid	37.5 mM	100	-	[103]
	Rhodosporidium fluviale DMKU-SP314	formic acid	0.5 g/L	100	-	[68]
	Rhodosporidium fluviale DMKU-SP314 Rhodosporidium toruloides Rhodosporidium toruloides Rhodosporidium toruloides	acetic acid formic acid acetic acid furfural	1.0 g/L 2, 4 g/L 5, 10, 20 g/L 1.0 g/L	72 25, 40 15.6, 50, 100 60	97 - -	[68]  [53]   [53]   [53]
	Rhodosporidium toruloides Y4 Mortierella isabelline DSM 1414 Mortierella isabelline NRRL 1757 Mortierella isabelline NRRL 1757	furfural furfural furfural 5-HMF	1 mM 21.8 mM 2.0 g/L 2.0 g/L	45.5 77 11 25	26.5 84 3 23	[101]  [20]  [104]  [104]
Lipid accumulation	Trichosporon fermentans CICC 1368	furfural	2.1, 4.7 mM		25, 50	[88]
	Trichosporon fermentans CICC 1368	HMF	15.1, 37.7 mM		25, 50	[88]

 

Point 8: The review does not provide a typical idea of ​​the material balance of the process of converting lignocellulose into lipids in comparison with the theoretical process in the absence of inhibitors. Therefore, the Conclusions are descriptive and lack the technical recommendations that the reader needs. This situation needs to be corrected.

Response 8: We appreciate the reviewer’s insightful comment regarding the need for a material balance comparison and technical recommendations in the conclusions. To address this, we have carefully revised the manuscript to incorporate:

• Page 9, Line 351-357: “Additionally, theoretical studies indicate that the maximum lipid yield from glucose metabolism can reach 32%, assuming all acetyl-CoA is directed toward lipid biosynthesis [65,66]. However, under practical fermentation conditions, metabolic losses, inhibitory effects, and process inefficiencies reduce actual lipid yields to approximately 22% [67]. In lignocellulose-derived hydrolysates, the presence of additional inhibitors further reduces conversion efficiency, leading to yields significantly below theoretical expectations [68].”
• Page 18, Line 709-718: “To improve conversion efficiency, future research should focus on optimizing bioreactor conditions, including controlled oxygen transfer, nutrient supplementation, and real-time process monitoring to dynamically adjust fermentation parameters. Additionally, detoxification strategies must be further refined to selectively remove inhibitors while preserving sugar integrity. Enhancing microbial strain resilience through metabolic engineering and adaptive evolution can further mitigate the inhibitory effects, allowing more efficient lipid accumulation. Finally, integrating pretreatment and fermentation strategies, such as sequential hydrolysis-fermentation approaches, can help balance sugar recovery and lipid productivity, ultimately making microbial lipid production more economically viable.”

 

Point 9: The review is very repetitive. Their exclusion would benefit the manuscript. Apparently, the review was written by several authors in separate sections. The text needs to be proofread and this situation corrected.

Response 9: We acknowledge the reviewer’s concern regarding redundancy in the manuscript. To address this, we have carefully proofread and revised the text to eliminate overlapping discussions while maintaining clarity, coherence, and completeness. The key revisions include:

• Page 4, Line 167-172: The sentences “Various methods are employed to pretreat lignocellulosic biomass for enhanced conversion. These include mechanical and biological approaches, along with alkaline pre-treatment and dilute acid hydrolysis. Processes such as ammonia fiber explosion and hydrothermal techniques, which encompass steam explosion and hot water treatment, are also included. Additionally, innovative green technologies like ionic liquids are emerging as promising alternatives in this area [17, 18].” have been deleted.
• Page 7, Line 270-273: The sentences “Cellulose is a linear polymer consisting only of D-glucose. Hemicellulose is a diverse polymer made up of various sugars like xylose and arabinose, as well as hexose sugars like mannose, glucose, and galactose, along with sugar-derived acids [47]” have been deleted.
• Page 13, Line 448-450: The sentences “By utilizing oxygen as an electron acceptor, laccase is capable of oxidizing phenolic compounds and generating byproducts. Laccase and peroxidase can effectively remove phenols from lignocellulosic hydrolysates.” have been deleted.
• Page 17, Line 653-654: The sentences “The inhibitors as discussed in the previous sections are the potent barriers hampering the microbial growth and thereby the microbial lipid production from lignocellulosic hydrolysates. Various inhibitors and their effects on oleaginous microbes and lipid production have been studied so far and discussed in this review.” have been revised: “Inhibitors remain a major challenge for microbial lipid production, requiring further research to enhance microbial tolerance and process optimization.”

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This review paper focuses on microbial lipids production from lignocellulosic biomass. The pretreatment approaches and reaction mechanisms of the conversion process are discussed. Several suggestions should be considered to enhance its quality.

Introduction: Clearly address the main novelty of this review paper compared to existing literature. Highlight what new insights readers can gain from it. It is suggested that the research gap should be described point by point in a separate section.

Please provide relevant information on significant research projects, especially recent large-scale ones worldwide on the relevant topic.

There is no sufficient information on the reactor design.

It is recommended that the reaction kinetics studies should be comprehensively examined and summarized, which is very important to improve the process conversion efficiency. Based on this, please discuss what challenges are facing ahead and how to improve these in the future.

Author Response

Point 1: Introduction: Clearly address the main novelty of this review paper compared to existing literature. Highlight what new insights readers can gain from it. It is suggested that the research gap should be described point by point in a separate section.

Response 1: Thank you for your suggestion. We have explicitly highlighted the novelty of this review in the Introduction section, emphasizing the unique aspects compared to existing literature：

Page 3, Line 114-122: “While microbial lipid production from lignocellulosic biomass has been reviewed extensively, most existing studies focus on general lipid biosynthesis pathways or broad fermentation strategies. In contrast, this review takes a more targeted approach by examining how inhibitors generated during biomass pretreatment affect microbial metabolism and lipid accumulation. In addition, it summarizes recent advances in detoxification strategies and fermentation optimization, which are crucial for improving process efficiency. By addressing these challenges and potential solutions, this review aims to provide a clearer perspective on how lignocellulose-based microbial lipid production can be further developed for large-scale applications.” was added.

We have now added a dedicated section summarizing the research gaps in a point-by-point format.

Page 3, Line 123-137: The section “Although microbial lipid production from lignocellulosic biomass has made significant progress, several challenges still hinder its large-scale application. One major issue is the presence of inhibitors such as furfural, hydroxymethylfurfural (HMF), and weak acids, which negatively impact microbial growth and lipid synthesis. While previous studies have identified these inhibitors, the exact metabolic and genetic mechanisms by which oleaginous microorganisms tolerate and adapt to them remain insufficiently understood. Additionally, various detoxification strategies have been explored, including chemical, enzymatic, and microbial approaches, but there is still no consensus on the most efficient and cost-effective method, particularly for industrial-scale applications. Another limitation is the difficulty in fully utilizing lignocellulosic hydrolysates, as microorganisms often exhibit low conversion efficiency when metabolizing mixed sugar substrates. This leads to suboptimal lipid yields and process inefficiencies. Furthermore, most studies on microbial lipid production are conducted at the laboratory scale, often in shake flasks or small bioreactors, and the transition to industrial-scale fermentation presents challenges related to oxygen transfer, agitation, and inhibitor removal.” was added.

 

Point 2: Please provide relevant information on significant research projects, especially recent large-scale ones worldwide on the relevant topic.

Response 2: Thank you for your valuable suggestion. Based on your suggestion, we have incorporated details of recent large-scale research projects related to lignocellulosic biomass conversion to microbial lipids.

Page 16, Line 642-648: There is a new paragraph about recent large-scale ones worldwide on the relevant topic. “Recent large-scale research initiatives have focused on overcoming economic and environmental challenges in microbial lipid production. The DOE Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) has been actively developing engineered yeast strains to enhance lipid accumulation and process efficiency in bio-based refineries. Additionally, studies on economic feasibility suggest that scaling up production to 48,000 tons per year could lower microbial lipid costs to $1.20/kg, making it competitive with conventional oils [135].”

 

Point 3: There is no sufficient information on the reactor design.

Response 3: Thank you for pointing this out. We have supplemented the manuscript with additional discussion on reactor design for microbial lipid production.

Page 17-18, Line 678-695: “In addition to strain engineering and inhibitor mitigation strategies, optimizing bioreactor design is another critical aspect of scaling up microbial lipid production. Reactor configurations, including batch, fed-batch, and continuous systems, influence key factors such as oxygen transfer, agitation, and inhibitor degradation, all of which play a fundamental role in microbial growth and lipid accumulation. Understanding the reaction kinetics in microbial lipid production using lignocellulosic hydrolysates is crucial for optimizing process efficiency. The presence of inhibitors in hydrolysates can significantly affect microbial metabolism, leading to reduced lipid yields. Studies have shown that the kinetics of microbial growth and lipid accumulation are influenced by factors such as substrate concentration, inhibitor presence, and oxygen availability. Developing kinetic models that accurately describe these interactions can aid in predicting process performance and designing effective bioreactor systems. For instance, incorporating inhibition kinetics into models can help in understanding how different pretreatment methods affect microbial activity and lipid production. Future research should focus on refining these models to account for the complex nature of lignocellulosic substrates and the dynamic conditions within bioreactors. Additionally, integrating realtime monitoring data with kinetic models could enable adaptive control strategies, enhancing overall process robustness and efficiency.”

 

Point 4: It is recommended that the reaction kinetics studies should be comprehensively examined and summarized, which is very important to improve the process conversion efficiency. Based on this, please discuss what challenges are facing ahead and how to improve these in the future.

Response 4: We appreciate this valuable suggestion. A new subsection has been added to provide a comprehensive summary of reaction kinetics studies in microbial lipid production. We have discussed the challenges ahead and how to improve them in the future:

Page 17, Line 683-695: “Understanding the reaction kinetics in microbial lipid production using lignocellulosic hydrolysates is crucial for optimizing process efficiency. The presence of inhibitors in hydrolysates can significantly affect microbial metabolism, leading to reduced lipid yields. Studies have shown that the kinetics of microbial growth and lipid accumulation are influenced by factors such as substrate concentration, inhibitor presence, and oxygen availability. Developing kinetic models that accurately describe these interactions can aid in predicting process performance and designing effective bioreactor systems. For instance, incorporating inhibition kinetics into models can help in understanding how different pretreatment methods affect microbial activity and lipid production. Future research should focus on refining these models to account for the complex nature of lignocellulosic substrates and the dynamic conditions within bioreactors. Additionally, integrating realtime monitoring data with kinetic models could enable adaptive control strategies, enhancing overall process robustness and efficiency.” We apologize that there is no related reference on the reaction kinetics. So, we have added the corresponding perspective on it.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The experimental article "Effects of lignocellulosic biomass-derived hydrolysates inhibitors on cell growth and lipid production during microbial fermentation of oleaginous microorganisms – A review" after the author's edits in response to the reviewer's recommendations has undergone major changes and is ready for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised paper has significantly improved.