Accumulation of Volatile Fatty Acids from Hydrothermally Treated Strawberry Extrudate through Anaerobic Fermentation at Different pH Values

Round 1

Reviewer 1 Report

The work is consistent with a number of other studies in the literature in which it is demonstrated that methanogenesis-inhibiting pH levels can favour acidogenesis other methane production, and may therefore be of eventual economic benefit to waste-generators. There will also be environmental benefits of moving away from methane production owing to a reduction in fugitive emissions from methane storage and transport infrastructures.

The work will be of interest to anyone researching this area, and adds to a growing understanding of acidogenesis from waste.

I have some issues however that will need updating before I believe the piece is suitable for publication.

(1) The word "recovery" in the title, and "obtaining" in the Abstract (line 21) implies that VFAs are being removed from the bioreactors, as opposed to simply accumulating/being produced within their liquid phases.

This distinction needs to be drawn because there are many studies now in which VFAs are being physically recovered from fermentation broths such as https://doi.org/10.1016/j.biortech.2021.125926

I also think that it would be useful to include comparisons of the economic value of methane and VFA in the introduction to help state the case for VFAs as a promising means through which to valorise waste.

I believe the word "concretely" throughout (5 instances, line 71 onwards) may be erroneous. I think the words "concurrently" or "similarly" may be more accurate.

I can't see the black bar for C2 on Figure 4b, and I can't see any vertical bards on Figure 4d

Suggest naming the acids on the legend of each Figure, rather than using C2, C3, etc, for consistency with prose

With these changes, I would recommend this work for publication

Author Response

General comments:

The work is consistent with a number of other studies in the literature in which it is demonstrated that methanogenesis-inhibiting pH levels can favour acidogenesis other methane production, and may therefore be of eventual economic benefit to waste-generators. There will also be environmental benefits of moving away from methane production owing to a reduction in fugitive emissions from methane storage and transport infrastructures.

The work will be of interest to anyone researching this area, and adds to a growing understanding of acidogenesis from waste.

I have some issues however that will need updating before I believe the piece is suitable for publication.

Reviewer´s comment 1: (1) The word "recovery" in the title, and "obtaining" in the Abstract (line 21) implies that VFAs are being removed from the bioreactors, as opposed to simply accumulating/being produced within their liquid phases.

 Authors´ response: both “recovery” and “obtaining” have been replaced by “accumulation” according to the reviewer comment (lines 1 and 21).

Reviewer´s comment 2: This distinction needs to be drawn because there are many studies now in which VFAs are being physically recovered from fermentation broths such as https://doi.org/10.1016/j.biortech.2021.125926.

Authors´ response: the authors agree with the reviewer about the adequacy of avoiding the use of recovery/obtaining terms, which can be confusing. Both terms have been removed from the manuscript. The modifications have been highlighted throughout the manuscript. Moreover, the journal article suggested by the reviewer has been included in the manuscript to improve the introduction section: “In fact, the economic value of the VFA is usually much higher than the economic value of the methane, typically obtained from anaerobic digestion processes (Jones et al., 2021). Actually, the methane has a market value of only €16.63-33.27/mt (EIA, 2022), whereas acetic acid and propionic acid had values of around €499/mt and €3546/mt, respectively (Petersen et al., 2018).” (lines 56-59). The new references have been also included in the References´ section.

Reviewer´s comment 3: I also think that it would be useful to include comparisons of the economic value of methane and VFA in the introduction to help state the case for VFAs as a promising means through which to valorise waste.

Authors´ response: The authors have included the information suggested by the reviewer in the following form: “In fact, the economic value of the VFA is usually much higher than the economic value of the methane, typically obtained from anaerobic digestion processes (Jones et al., 2021). Actually, the methane has a market value of only €16.63-33.27/mt (EIA, 2022), whereas acetic acid and propionic acid had values of around €499/mt and €3546/mt, respectively (Petersen et al., 2018).” (lines 56-59).

Reviewer´s comment 4: I believe the word "concretely" throughout (5 instances, line 71 onwards) may be erroneous. I think the words "concurrently" or "similarly" may be more accurate.

Authors´ response: The authors have removed the word “concretely” from the manuscript.

Reviewer´s comment 5: I can't see the black bar for C2 on Figure 4b, and I can't see any vertical bards on Figure 4d

Authors´ response: The authors apologize for the mistake. It was an error in the conversion from word to pdf format. The error has been solved and the Figure 4 can be properly seen in the revised version.

Reviewer´s comment 6: Suggest naming the acids on the legend of each Figure, rather than using C2, C3, etc, for consistency with prose

Authors´ response: The authors have replaced the legends to ensure the consistency in the use of terms for naming the acids.  (See figure 4)

Reviewer´s comment 7: With these changes, I would recommend this work for publication

 Authors´ response: the authors´ acknowledge the reviewer by his/her valuable contribution to the manuscript.

Reviewer 2 Report

Manuscript Number: agronomy-2055694-peer-review-v1

Title: Recovery of volatile fatty acids from hydrothermally treated strawberry extrudate through anaerobic fermentation at different pH values

 Line 30-32 on Page 1: " A stream enriched in acetic acid was obtained at pH 9, whereas the operation at pH 5 resulted in a more complex composition with a high percentage of propionic acid." What is the ratio or concentration of acetic acid and propionic acid under different pretreatment conditions？

 What are the advantages of the combination of hydrothermal pretreatment and pH control?

 Page 9-11: Figure 4 does not match the corresponding text description.

Author Response

Title: Recovery of volatile fatty acids from hydrothermally treated strawberry extrudate through anaerobic fermentation at different pH values

Reviewer´s comment 1: Line 30-32 on Page 1: "A stream enriched in acetic acid was obtained at pH 9, whereas the operation at pH 5 resulted in a more complex composition with a high percentage of propionic acid." What is the ratio or concentration of acetic acid and propionic acid under different pretreatment conditions?

Authors´ response: This information has been included in the abstract according to the reviewer´s suggestion in the following form: “A stream enriched in acetic acid was obtained at pH 9 (around 65% of the VFA), whereas the operation at pH 5 resulted in a more complex composition with a high percentage of propionic acid (29% of the VFA).” (lines 31-33)

Reviewer´s comment 2: What are the advantages of the combination of hydrothermal pretreatment and pH control?

Authors´ response: A sentence clarifying the interest of combining both strategies has been included in the introduction section: “Despite the limitation on the hydrolysis rates, the operation at acidic or alkaline pH allowed an effective inhibition of the methanogenesis and the conversion of most of the soluble organic matter into VFA [13].

Due to the limited solubilization reported for the SE, it would be interesting to evaluate a pre-treatment method that would facilitate the hydrolysis of the substrate and, thus, improve the process yield. The implementation of hydrothermal treatments has been widely proposed for enhancing the hydrolysis of lignocellulosic substrates [6,17].” (lines 72-78).

Reviewer´s comment 3: Page 9-11: Figure 4 does not match the corresponding text description.

Authors´ response: The figure description has been modified to make it more descriptive of the Figure 4: “Figure 4. Individual VFA compounds, i.e. acetic acid, propionic acid, butyric acid and valeric acid, accumulated for a) pH5 SE, b) pH5 HTSE, c) pH 9 SE, and d) pH9 HTSE, against the accumulated added substrate (added COD_tot) throughout the experimental time.” (lines 253-256)

Reviewer 3 Report

This work investigated the effects of pH values on volatile fatty acids from hydrothermally treated strawberry extrudate through anaerobic fermentation. However, the novelty of this work was lacked since the impacts of pH on substrates fermentation has been widely investigated. Also, some comments should be addressed.

1. Why do authors choose pH 5 and pH 9? Why do not investigate the other pH, e.g., pH 6, 7, 8?

2. The authors investigated the efficiency of hydrolysis, acidification, and methanogenesis based on the calculation of COD. However, why authors do not investigate the indicators of these stages (e.g., soluble NH4+-N, hydrolytic and acidogenic enzymatic activity, F420)

3 Why do authors did not investigate the variation of microbial structure, since the anaerobic fermentation is mainly a microbial-driven process

Author Response

General comment:

This work investigated the effects of pH values on volatile fatty acids from hydrothermally treated strawberry extrudate through anaerobic fermentation. However, the novelty of this work was lacked since the impacts of pH on substrates fermentation has been widely investigated. Also, some comments should be addressed.

Reviewer´s comment 1: Why do authors choose pH 5 and pH 9? Why do not investigate the other pH, e.g., pH 6, 7, 8?

Authors´ response: As it was already stated in the original version of the manuscript, the pH was selected to avoid favourable conditions for the methanogenic activity. The range where the methanogenic activity can occur has been updated according to the reviewer´s comment: “The anaerobic reactor of the pH-controlled anaerobic fermentation operates at pH values different from 6-8 that is recommended for methanogenic activity [15], avoiding the conversion of the solubilised compounds into biogas and, thus, favouring the accumulation of VFA.” (lines 59-61).

Reviewer´s comment 2: The authors investigated the efficiency of hydrolysis, acidification, and methanogenesis based on the calculation of COD. However, why authors do not investigate the indicators of these stages (e.g., soluble NH4+-N, hydrolytic and acidogenic enzymatic activity, F420).

Authors´ response: We appreciate the comment of the reviewer. Although the additional information that would be provided by the monitoring of these indicators would be very illustrative, the monitoring of the soluble COD, the VFA and the methane production allowed the expression of all the measurements as COD and, thus, the elaboration of balances to easily compare the relation of the different stages at each experimental condition.

Reviewer´s comment 3: Why do authors did not investigate the variation of microbial structure, since the anaerobic fermentation is mainly a microbial-driven process

Authors´ response: The absence of organic matter wash-out in a batch system can strongly limit the accuracy of the measurements of the microbial structure by, for example, combination of 16S and qPCR. DNA from microorganisms in the reactors that would be identified even if they are not active due to the experimental conditions and, thus, the obtained results would not be representative of the effect of the experimental conditions over the microbial activity.