Review Reports - Towards Higher Energy Conversion Efficiency by Bio-Hydrogen and Bio-Methane Co-Production: Effect of Enzyme Loading and Initial pH

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript: fermentation-3801295 Towards higher energy conversion efficiency by bio-hydrogen and bio-menthane co-production: effect of enzyme loading and initial pH

Subject: Hydrogen production through photo-fermentation, along with an enzyme strategy, is associated with methane production from anaerobic digestion.

Abstract: TS - Please clarify the meaning at the beginning.
Introduction: “Its capacities to use various (several) sources (for example, …..) of feedstocks”; As enzymes are used to enhance conversion efficiency, authors are encouraged to discuss the associated costs in the introduction section. Previous studies have explored the role of enzymes and pH in hydrogen production. Please clarify the novel contributions of your research.
Material and methods: Replace Lux by lx
It's important to note that the results obtained by the authors align with findings from previous studies in literature, particularly regarding the variable profile. For instance, a notable decline in productivity is observed after 24 to 48 hours of photo-fermentation. It would be beneficial to consult and cite at least two references that address and discuss this challenge (https://doi.org/10.1016/j.ijhydene.2025.150088 and https://doi.org/10.3390/fermentation10040213.

Author Response

Dear Reviewer,

Thank you very much for your thoughtful review and constructive comments on our manuscript titled “Towards higher energy conversion efficiency by bio-hydrogen and bio-menthane co-production: effect of enzyme loading and initial pH” (Manuscript ID: fermentation-3801295). We greatly appreciate the time and effort you have dedicated to providing valuable feedback, which has helped us improve the quality of our work. Below, we have addressed each of your comments point by point and made the necessary revisions to the manuscript.

Point 1. TS - Please clarify the meaning at the beginning.

Response to Point 1: Thanks for your comments, we have represented the abbreviation for TS with the full name Total Solid in the revised version.

Point 2. Introduction: "Its capacities to use various (several) sources (for example, ……) of feedstocks"; As enzymes are used to enhance conversion efficiency, authors are encouraged to discuss the associated costs in the introduction section. Previous studies have explored the role of enzymes and pH in hydrogen production. Please clarify the novel contributions of your research.

Response to Point 2: Thank you for your careful review. We have added discussions on hydrogen production costs in the introduction, with the relevant content highlighted in red. The novelty of this study lies in integrating photo-fermentative biohydrogen production with methanogenesis to maximize energy conversion efficiency of duckweed. Additionally, we systematically analyzed the interactive effects of enzyme loading and pH on biohydrogen-methane co-production, overcoming the limitations of single-parameter optimization or optimization confined to a single stage. Biohydrogen-methane co-production mode enables stepwise energy recovery from duckweed, validating the efficiency and practicality of the technical pathway and providing data support for large-scale applications.

Point 3. Material and methods: Replace Lux by lx

Response to Point 3: Thanks for your careful review. We have replaced "Lux" with "lx" in the Material and methods section and marked the change with red color in the revised version.

Point 4. It’s important to note that the results obtained by the authors align with findings from previous studies in literature, particularly regarding the variable profile. For instance, a notable decline in productivity is observed after 24 to 48 hours of photo-fermentation. It would be beneficial to consult and cite at least two references that address and discuss this challenge. (https://doi.org/10.1016/j.ijhydene.2025.150088 and https://doi.org/10.3390/fermentation10040213.

Response to Point 4: Thank you very much for your insightful suggestions. In this study, a significant decline in biohydrogen production rate was observed after 24 - 48 hours. Following your advice, we carefully reviewed two references: Gabriela Aparecida Santos et al. (https://doi.org/10.1016/j.ijhydene.2025.150088) systematically analyzed the metabolic kinetics of photo-fermentative biohydrogen production, specifically noting that substrate depletion and accumulation of inhibitory metabolites (e.g., organic acids) cause a marked decrease in biohydrogen yield. Mélida del Pilar Anzola-Rojas et al. (https://doi.org/10.3390/fermentation10040213) revealed that reduced activity of key biohydrogen-producing bacteria constitutes another critic al factor contributing to the yield decline. These findings support our experimental results. In the revised manuscript, we have added a discussion on the consistency between the observed decline in hydrogen production rate and previous studies, along with a detailed elaboration of the underlying mechanisms emphasized in these references.

We believe that the revisions have significantly improved the clarity, rigor, and overall quality of the manuscript. We are grateful for the reviewer's insightful comments, which have helped us strengthen our work. If there are any further clarifications or additional revisions needed, please do not hesitate to let us know. Thank you once again for your time and valuable feedback.

Reviewer 2 Report

Comments and Suggestions for Authors

Compared to the control group (blank group), how much can bio-hydrogen and bio-methane be increased?
The conclusion and abstract are almost identical, and the main content mostly presents research data and results, lacking important arguments and discussions.
Although duckweed has undergone ash analysis, it would be easier to analyze and discuss if there were analyses of cellulose, hemicellulose, lignin, and sugar components.
Were there any vibrations in photo fermentation biohydrogen production (Phase I) ? And Phase II?
Results and discussion: In principle, the text should precede the charts and figures.
Figure 2.: At the 12-hour monitoring shows sugar present. Where does the sugar come from? How long does it take for sugar to form after adding the enzyme? What happens during the 0-12 hour period?
The effect of different enzyme loading combined with pH on time-change profile of 296 pH during the bio-CH4 production process is present in Fig. 3b, 3d, and 3f. However, Simply based on different enzyme loading and pH, it is impossible to determine what factors are influencing bio-CH4 production. It is still necessary to investigate each factor individually through product analysis and biochemical engineering.
3. Effect of different enzyme loadings combined with initial pH on soluble fermentation metabolites during biohydrogen-biomethane co-production process: The process of producing hydrogen does not discuss how acetic acid, butyric acid, and ethanol are generated. Similarly, the process of producing methane does not discuss the relationship between acetic acid, butyric acid, and ethanol and the generation of methane.
It was found that the initial pH of 8.0 achieved the highest energy conversion efficiency over the other initial pH values no matter the enzyme loadings during the bio-H2 production process. Why?

Author Response

Dear Reviewer,

Point 1. Compared to the control group (blank group), how much can bio-hydrogen and bio-methane be increased?

Response to Point 1: Thanks for your careful review. Under the optimal condition (30% enzyme loading + pH 8.0) in the photo-fermentative bio-hydrogen and bio-methane co-production mode, the bio-methane yield (260.32 mL/g TS) in the anaerobic methanogenesis stage increased by 397.65% compared with the control group (52.31 mL/g TS).

Point 2. The conclusion and abstract are almost identical, and the main content mostly presents research data and results, lacking important arguments and discussions.

Response to Point 2: Thanks for your comments. This is a valuable critique that will significantly strengthen our manuscript. We have revised the abstract and conclusion according to your suggestions, and the modified content has been highlighted in red.

Point 3. Although duckweed has undergone ash analysis, it would be easier to analyze and discuss if there were analyses of cellulose, hemicellulose, lignin, and sugar components.

Response to Point 3: We sincerely appreciate your insightful suggestion and fully agree with them. Current research focuses on ash analysis and elemental composition, but we recognize that analyzing organic components (cellulose, hemicellulose and lignin) can strengthen process interpretation. Therefore, we have supplemented detailed data on cellulose, hemicellulose and lignin in duckweed in the "Materials and Methods" section of the revised version, and marked them in red. We monitored changes in reducing sugar concentration during photo-fermentation biohydrogen production. On the one hand, measuring reducing sugars concentration focuses on the core substrate of the biological reaction, effectively reflecting sugar metabolic dynamics. On the other hand, the detection process is more convenient and rapid compared to determining sugar components. Nevertheless, we still believe that your suggestion is highly beneficial for our subsequent research, sugar components analysis may enable us to gain a deeper understanding of the compositional changes throughout the process.

Point 4. Were there any vibrations in photo fermentation biohydrogen production (Phase I)? And Phase II?

Response to Point 4: We sincerely appreciate your valuable suggestion. In the current experimental design, both the photo-fermentation bio-hydrogen production stage (Phase I) and the methane production stage (Phase II) were conducted under static conditions. However, your innovative proposal on vibration has inspired a new research direction. We plan to introduce agitation in future studies to systematically investigate the effects of different vibration intensities and modes (e.g., intermittent or continuous) on bacterial distribution, light utilization efficiency, and hydrogen yield in the photo-fermentation system, as well as their impacts on mass transfer and microbial cooperation in the second-stage methanogenesis. This innovative proposal provides substantial guidance for advancing our subsequent research initiatives, which significantly contributes to the advancement of sustainable bioenergy production technologies. We sincerely thank you for this inspiration and will implement this idea in subsequent research for validation.

Point 5. Results and discussion: In principle, the text should precede the charts and figures.

Response to Point 5: Thank you for your careful review. We fully agree with the principle in the "Results and Discussion" section that "the text should precede before the charts and figures". In the revised manuscript, we have accordingly adjusted the layout. This has further enhanced the readability and coherence of the article. Once again, we thank you for your valuable suggestions in improving the manuscript.

Point 6. Figure 2: At the 12-hour monitoring shows sugar present. Where does the sugar come from? How long does it take for sugar to form after adding the enzyme? What happens during the 0-12 hour period?

Response to Point 6：Thank you for your careful review. The reducing sugars quantified at 12 h were mainly generated by enzymatic depolymerization of structural polysaccharides in duckweed biomass, specifically cellulose (β-1,4-glucan) and hemicellulose (xylan/glucomannan). To our knowledge, the initiation time of enzymatic saccharification (i.e., when sugars can detectable) closely linked to reaction conditions such as temperature and pH. During the initiation phase (0-2 h) of enzymatic hydrolysis, cellulase adsorbed onto the substrate surface, generating trace amounts of sugars; as hydrolysis accelerated, substantial quantities of sugars were produced in the reaction system. Given that a simultaneous saccharification approach was employed in this study, the reducing sugar concentration at 12 h reflects the dynamic balance between enzyme-catalyzed sugar production and microbial sugar consumption, representing a state of production-consumption equilibrium rather than the endpoint of hydrolysis reaction. Furthermore, photo-fermentation bio-hydrogen production is a complex system involving multiple reactions, during 0-12 h period includes, alongside the primary substrate enzymatic hydrolysis, processes such as bacterial growth and metabolism, sugar/acid conversion, and light-driven electron transfer.

Point 7. The effect of different enzyme loading combined with pH on time-change profile of 296 pH during the bio-CH₄ production process is present in Fig. 3b, 3d, and 3f. However, simply based on different enzyme loading and pH, it is impossible to determine what factors are influencing bio-CH₄ production. It is still necessary to investigate each factor individually through product analysis and biochemical engineering.

Response to Point 7: We sincerely appreciated your insightful comments. The main objective of this study was to investigate the impact of effluent generated from photo-fermentation bio-hydrogen production under different process conditions (enzyme loading and initial pH) on methanogenesis, thereby enhancing duckweed’s energy conversion efficiency via bio-hydrogen and bio-methane co-production. Additionally, Figure 4a presented the composition of terminal soluble metabolites in the fermentation broth under different process conditions, and sought to clarify the impact of bio-hydrogen production parameters on methanogenesis from the perspective of metabolites. Finally, we fully agree that your suggestion to explore the impact of individual process parameters on methanogenesis in co-production is highly valuable, and we will pursue this in subsequent studies.

Point 8. Effect of different enzyme loadings combined with initial pH on soluble fermentation metabolites during biohydrogen-biomethane co-production process: The process of producing hydrogen does not discuss how acetic acid, butyric acid, and ethanol are generated. Similarly, the process of producing methane does not discuss the relationship between acetic acid, butyric acid, and ethanol and the generation of methane.

Response to Point 8: Thank you for your careful review. In Section 3.3 of Results and Discussion, the production process of ethanol has already been described and highlighted in red. In this study, our discussion focuses on the composition of acetic acid, butyric acid, and ethanol, as well as the correlation between process conditions and the composition of liquid-phase end products. It is well known that during the hydrogen production process, the sugars derived from the enzymatic hydrolysis of duckweed will be decomposed by microbial metabolic pathways into acetic acid, butyric acid, and ethanol, accompanied by the generation of energy (ATP), as shown in Equations (1)-(3). It is worth noting that the acidification stage in the methane production process is consistent with the reaction process in the biohydrogen production. During the methanogenesis stage, methanogenic bacteria not only can efficiently utilize the products from the acidification stage, but also can use the effluent from the hydrogen production process as a substrate, ultimately converting into CH₄ and CO₂. Among them, acetic acid can be directly utilized by methanogenic bacteria; while butyric acid and ethanol need to be first degraded by symbiotic bacteria into products such as acetic acid (as shown in Equations (4) - (5)), and then used by anaerobic bacteria for methane production Equations (6).

C₆H₁₂O₆+ 2H₂O → 2CH₃COOH + 2CO₂+ 4H₂+ 2ATP (1)

C₆H₁₂O₆→ CH₃CH₂CH₂COOH + 2CO₂+ 2H₂+ 3ATP (2)

C₆H₁₂O₆→ 2CH₃CH₂OH+2CO₂+ 2ATP (3)

CH₃CH₂OH + H₂O → CH₃COOH + 2H₂ (4)

CH₃CH₂CH₂COOH + 2H₂O → 2CH₃COOH + 2H₂(5)

CH₃COOH → CH₄+ CO₂(6)

Point 9. It was found that the initial pH of 8.0 achieved the highest energy conversion efficiency over the other initial pH values no matter the enzyme loadings during the bio-H2 production process. Why?

Response to Point 9: Thank you very much for your careful review. In the biohydrogen production process, the initial pH of 8.0 consistently achieved the highest energy conversion efficiency regardless of enzyme loadings, and this phenomenon can be attributed to the following factors: 1) The dominant hydrogen-producing bacteria (e.g., Rhodobacter) in the photo-fermentative hydrogen production system exhibit optimal growth and metabolic activity under weakly alkaline conditions (pH 7.5–8.5). This may be associated with the similarity to the growth environment of photosynthetic bacteria in their culture medium. Consequently, at pH 8.0, the bacterial cell membrane integrity, enzyme synthesis capacity (such as hydrogenase and nitrogenase), and electron transport chain efficiency are all enhanced, increasing the biohydrogen yield. 2) Under weakly alkaline conditions, the accumulation of volatile fatty acids is alleviated, thereby avoiding the inhibition of microbial activity. In summary, the initial pH of 8.0 optimizes microbial physiological status, enzyme activity, and metabolite balance, making it the optimal condition for achieving the highest energy conversion efficiency in this biohydrogen production system.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors The notation '3000lx' should be revised to '3,000lx' throughout the manuscript. Ensure consistency in all similar instances prior to publication. After that, the manuscript can be published. Comments on the Quality of English Language The notation '3000lx' should be revised to '3,000lx' throughout the manuscript. Ensure consistency in all similar instances prior to publication. After that, the manuscript can be published.

Author Response

Dear Reviewer,

Thank you very much for your thoughtful review of our manuscript entitled "Towards Higher Energy Conversion Efficiency through Co-production of Biohydrogen and Biomethane: Effects of Enzyme Loading and Initial pH" (Manuscript ID: Fermentation-3801295). We greatly appreciate the time and effort you have dedicated to providing valuable feedback, which has been instrumental in helping us enhance the quality of our work. Below, we have carefully addressed each of your comments and incorporated the necessary revisions into the manuscript.

Point 1. The notation '3000lx' should be revised to '3,000lx' throughout the manuscript. Ensure consistency in all similar instances prior to publication. After that, the manuscript can be published.

Response to Point 1: Thank you very much for your careful review and valuable suggestion. We have revised all instances of '3000lx' to '3,000lx' throughout the manuscript and thoroughly checked other similar numerical notations to ensure consistency in their formatting. The revised manuscript now adheres to the required standards.

We are deeply grateful for your careful review, and once again, we thank you for taking the precious time and providing valuable feedback.

Reviewer 2 Report

Comments and Suggestions for Authors

No more comments.

Author Response