Rumen-Derived Consortia Shaped by Substrate-Specific Enrichment Show Specialized Lignocellulose Utilization, Diversified Hydrogen Metabolism, and Cryopreservation Stability

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

The manuscript investigates the development of substrate-adapted rumen microbial consortia through sequential enrichment on structurally distinct substrates (alfalfa, barley straw, CMC, and xylan). The study combines fermentation analysis, microbial community profiling, and cryopreservation assessment to evaluate the stability and functional specialization of enriched consortia.

The topic is relevant and timely because manipulation of the rumen microbiota to improve feed efficiency and reduce methane emissions is an important research area in animal nutrition and microbial ecology. The manuscript provides interesting observations regarding substrate-driven microbial selection, volatile fatty acid (VFA) shifts, and potential hydrogen sink pathways.

Major Comments

1. Overinterpretation of Hydrogen Metabolism and Methane Reduction

One of the major weaknesses of the manuscript is the strong emphasis on hydrogen redirection and methane mitigation without directly measuring methane production, hydrogen fluxes, archaeal populations, or reductive metabolic pathways. The authors repeatedly suggest that substrate-enriched consortia may reduce methanogenesis by redirecting reducing equivalents toward propionate or caproate production. However, no direct measurements of methane emissions, dissolved hydrogen, archaeal abundance, or metagenomic/metatranscriptomic evidence supporting these mechanisms were provided. The discussion should be substantially toned down, and the authors should clearly indicate that these interpretations based only on indirect metabolic indicators.

2. Clarification Needed for Substrate Composition

More detailed information is needed regarding the chemical characteristics of the substrates used in the enrichment experiments. This information is essential for understanding substrate-driven microbial selection and fermentation patterns.

The authors should provide detailed information about chemical composition of substrates,

3. Figure

Figure 2 require improvement in terms of readability.

4. Absence Conclusion Section

 

Author Response

 Reviewer 1 Major Comments

1. Overinterpretation of Hydrogen Metabolism and Methane Reduction

One of the major weaknesses of the manuscript is the strong emphasis on hydrogen redirection and methane mitigation without directly measuring methane production, hydrogen fluxes, archaeal populations, or reductive metabolic pathways. The authors repeatedly suggest that substrate-enriched consortia may reduce methanogenesis by redirecting reducing equivalents toward propionate or caproate production. However, no direct measurements of methane emissions, dissolved hydrogen, archaeal abundance, or metagenomic/metatranscriptomic evidence supporting these mechanisms were provided. The discussion should be substantially toned down, and the authors should clearly indicate that these interpretations based only on indirect metabolic indicators.

Response: We would like to express our sincere gratitude to the reviewers for their insightful and constructive feedback regarding our manuscript, "microorganisms-4296661." We found the comments instrumental in refining the scientific rigor and clarity of our work.

We agree that the original text required a more cautious interpretation. Consequently, we have "toned down" the discussion in lines 490-520(Implications for hydrogen metabolism )

. Definitive claims regarding methanogenesis inhibition have been replaced with probabilistic and interpretative language, clarifying that these shifts are suggested by metabolic byproducts (VFAs) rather than direct flux measurements. Discussion is toned down to  acknowledge that direct measurements of methane or dissolved hydrogen were not performed. We have revised the text to categorize propionate and caproate as "potential sinks" for reducing equivalents that compete with hydrogenotrophic methanogens. Modified phrasing to include "suggests," "potential," and "likely reflects," while explicitly acknowledging the lack of direct methane measurement.

 

1. Clarification Needed for Substrate Composition

More detailed information is needed regarding the chemical characteristics of the substrates used in the enrichment experiments. This information is essential for understanding substrate-driven microbial selection and fermentation patterns.

The authors should provide detailed information about chemical composition of substrates,

Response: We have included chemical composition descriptions of all substrates, distinguishing between monocots/dicots and specifying the sources for recalcitrant fractions. (Lines 84–90) Inserted detailed chemical composition and its recalcitrance value.

1. Figure

Figure 2 require improvement in terms of readability.

Response: We thanks the reviewer for the suggestion to improve the readability of the figure. In response, we have revised the visualization to make it clearer and easier to interpret. The updated figure now uses a more intuitive color scheme to distinguish experimental groups, improving visual separation and reducing confusion from overlapping information. We have also enhanced the overall layout by increasing the clarity of axis labels and text elements so that trends can be more easily followed across VFAs and substrates. In addition, we improved the presentation of statistical significance indicators to ensure they are more clearly visible and do not overlap with the data. These changes collectively improve the clarity, interpretability, and overall visual accessibility of the figure, making the biological patterns easier to understand. New figure legend added.

1. Absence Conclusion Section

Response: Conclusion section added Line 571-580.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a highly relevant and timely topic by investigating rumen-derived microbial consortia enriched under different substrate conditions and their functional specialization in lignocellulose degradation and hydrogen metabolism. The study has clear potential to contribute to our understanding of rumen microbial ecology and its application in improving fiber utilization and reducing methane emissions. The integration of substrate-driven enrichment, metabolic profiling, and cryopreservation stability is particularly valuable. However, several aspects of experimental design, data interpretation, and biological contextualization require clarification. In particular, the linkage between observed microbial shifts and functional outcomes (hydrogen metabolism pathways and lignocellulose degradation efficiency) should be more explicitly supported. Overall, the manuscript is promising but would benefit from minor revisions to improve clarity, consistency, and biological interpretation.

 

Introduction:

-Abbreviations should be defined at first mention and used consistently throughout the manuscript.

-The practical implications of substrate-specific enrichment for animal nutrition (feed digestibility and methane mitigation) should be more explicitly clarified and better linked to the study findings.

Materials and Methods:

-The substrate composition, particularly the lignocellulosic structure, should be described in greater detail.

-The cryopreservation procedure (including cryoprotectant, storage duration, and thawing conditions) should be described more clearly and in greater detail.

-The results related to hydrogen metabolism should be more clearly categorized (distinguishing hydrogenotrophic vs. fermentative pathways).

Conclusion:

-There is excessive generalization throughout the manuscript. The application potential should be expressed more cautiously and with appropriate limitations.

Author Response

Reviewer 2 : The manuscript addresses a highly relevant and timely topic by investigating rumen-derived microbial consortia enriched under different substrate conditions and their functional specialization in lignocellulose degradation and hydrogen metabolism. The study has clear potential to contribute to our understanding of rumen microbial ecology and its application in improving fiber utilization and reducing methane emissions. The integration of substrate-driven enrichment, metabolic profiling, and cryopreservation stability is particularly valuable. However, several aspects of experimental design, data interpretation, and biological contextualization require clarification. In particular, the linkage between observed microbial shifts and functional outcomes (hydrogen metabolism pathways and lignocellulose degradation efficiency) should be more explicitly supported. Overall, the manuscript is promising but would benefit from minor revisions to improve clarity, consistency, and biological interpretation.

 

Introduction:

-Abbreviations should be defined at first mention and used consistently throughout the manuscript.

Response: All abbreviations are now defined at first mention

-The practical implications of substrate-specific enrichment for animal nutrition (feed digestibility and methane mitigation) should be more explicitly clarified and better linked to the study findings.

Response: we have explicitly linked substrate-specific enrichment to critical practical outcomes, specifically focusing on the drivers of feed efficiency and the mitigation of enteric methane emissions.

Introduction-Line 59-62:Therefore developing rumen-derived probiotic consortia that can efficiently of targeting specific, recalcitrant feed fractions while partition hydrogen away from methane toward beneficial volatile fatty acids (VFAs) potentially may enhance the feed efficiency.

Line 532-535: These specialized consortia provide a model for exploring how tailored microbial networks might target recalcitrant feed fractions or serve as alternative hydrogen sinks, though their theoretical role in methane mitigation requires confirmation through direct flux measurements

Line 546-550:The stability of these cross-feeding networks presents a potential avenue for developing consortia that might help stabilize fermentation during weaning transitions. While their ecological compatibility is promising, their ability to prevent metabolic disorders and support animal performance is a hypothesis that requires further testing in live animals

 

 

Materials and Methods:

-The substrate composition, particularly the lignocellulosic structure, should be described in greater detail.

Response: We have included chemical composition descriptions of all substrates, distinguishing between monocots/dicots and specifying the sources for recalcitrant fractions. (Lines 84–90) Inserted detailed chemical composition and its recalcitrance value.

 

-The cryopreservation procedure (including cryoprotectant, storage duration, and thawing conditions) should be described more clearly and in greater detail.

Response:

Detailed Cryopreservation Protocol rewritten Line 141-152, to improve clarity and provided greater details : We have specified that 6 mL of culture was mixed with MC− medium and 15% glycerol (final concentration) in 2-ml cryogenic vials, followed by flash-freezing in liquid nitrogen (Lines 134–141). Storage duration (one months). We have added the specific detail that samples were thawed at 20°C under CO₂ to ensure the recovery of viable anaerobic populations.

 

-The results related to hydrogen metabolism should be more clearly categorized (distinguishing hydrogenotrophic vs. fermentative pathways).

Response: We thank the reviewers for these critical insights. We have substantively revised Section. Line 490-498; “Implications for Hydrogen Metabolism”, we have introduced a clear categorization of the metabolic results, distinguishing between "fermentative -evolving pathways" and "reductive -consuming (hydrogenotrophic) pathways" to provide better structural and biological clarity

 

 

Conclusion:

-There is excessive generalization throughout the manuscript. The application potential should be expressed more cautiously and with appropriate limitations.

 

Response: To address the concern regarding excessive generalization, we have systematically revised the manuscript—including the Abstract, Introduction, and Discussion—to shift from definitive claims to a probabilistic and interpretative tone. We replaced definitive verbs with more cautious phrasing, such as "suggests a potential," "theoretically," and "could potentially," to clarify that the observed metabolic shifts are preliminary interpretations based on indirect VFA indicators rather than direct measurements of hydrogen or methane flux. Furthermore, we have explicitly acknowledged methodological limitations by inserting disclaimers in the Discussion and Conclusion, emphasizing that while these specialized consortia provide a functional model for energy redirection, their practical in vivo impact remains a hypothesis that requires confirmation through direct flux measurements. By also categorizing metabolic pathways into "fermentative -evolving" and "reductive -consuming" (hydrogenotrophic) routes, we have replaced broad generalizations with a more precise biological framework that avoids over-claiming application potential while accurately reflecting the scope of our data

 

 

 

 

 

 

 

 

Author Response File: Author Response.pdf