Aquaculture Sludge as Co-Substrate for Sustainable Olive Mill Solid Waste Pre-Treatment by Anthracophyllum discolor

Round 1

Reviewer 1 Report

Presented manuscript concerns pre-treatment of olive oil solid waste using A. discolor supported by aquaculture sludge in order to remove phenols and produce enzymes. Manuscript falls within the Journal scope, might be interesting, however, improvements are required before further processing of this manuscript. For this reason see the comments below.

Section. 2.4. Please provide more details how laccase activity was determined.

Please explain how activity was calculated for various enzymes, when teh same subsrate was used?

In the whole manuscript there is a lack of deeper explanantions of the obtained data. In fact only data presentataion and its discussion with previosly published studies is presented. It has to be improved in the whole manuscript.

L285-L286. Something is wrong with sentence presented in these lines.

L269-275. This sentence is too long, too complicated and oveestimated and should be rewritten.

In the whole manuscript there is a lot of editorial, grammar and syntax issues. It should be improved.

Some data should be added in the Conclusions section to support preseted statements.

Minor improvements of References list are required.

Author Response

Thank you for your comments 

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Lines 25: the phenol and lignin removal

Line 26: OMWS supplemented with Aquaculture sludge (AS) as an external nitrogen source by Anthracophyllum discolor.

Line 27: Consider “the “highest” instead of “the best” and “were” instead of “was.”

Line 28: Consider “In these conditions” instead of “under the same conditions.”

Line 29: 56%of what? (Lignin? Phenol?)

Line 33: Delete comma after “activity.”

Lines 44-45: Rephrase and change ref 4, since these are about liquid waste (OMWW).

Line 54: Omit “method”. “White-rot fungi” all caps or all lower-case letters.

Line 65: Replace “dephenolate” with “reduce the phenolic content of”

Line 69: Replace “good” with “high.”

Line 152: Omit “activity”

Line 94: Consider replacing “the bread corresponds to Arbequina” with “The processed olives belonged to Arbequina variety.”

Lines 102-103: Explain what is the solid and liquid phase of the biological pre-treatment (or omit the phrase). Instead of the comma add “and were.”

Line 104: Delete “For”

Line 107: Delete “For”

Line 112: that was used

Line 116: “cultivated” instead of “performed”

Line 119: Kirk’s medium

Line 134: what is this “dry mixture”? Do you mean dry OMSW? Explain.

Line 136: Consider “control” instead of “blank”

Line 135: explain what “destructive manner” means.

The description of the experimental design is not clear. More details are needed. For example you should describe in detail the “dry basis mass OMSW (g)”. You added a “culture medium” as it is shown in Table 1, and you don’t mention it at all in the text that describes table1. Is it Total Solids? Then replace the “culture medium concentration” the “culture medium TS”. If you mean the “mixture” with the term “culture medium” then replace it. Use one term for same treatment in tables as well as in the text: (culture medium or pre-treatment or mixture if these are the same). Give more details about how you prepared the various mixtures.

Line 140: were “incubated” instead of “carried out”

Line 141:and at the end

Line 141: Table 1 is included in table 2 (except for OMWS and AS which are used as a control). If so, unify your results into a single table.

Many of the “pre-treatments” in Table 1 and 2 have the same name but they are different. Give different names for different “pre-treatments”.

Line 152: Delete “activity”

Line 160-161: Give the full name of the enzymes, at least once in the text.

Line 181: Give the number of different pre-treatment replications (n=?).

Line 186: reported

Line 189: where are these results? (In table 3, TSP measurements are missing).

Line 189: what is AG?

Line 196: recover

Line 199: Consider rephrasing “The nitrogen content of AS was 7.92%,”

Line 205: instead

Line 212: reported

Figure 1b: Consider rescaling the y-axis to 0-50% and place the error bars (if any).

Line 239: Give the reference number

Line 247: Do you mean higher (instead of significant)?

Lines 248-249: Supplementary data Figure 2 are missing. Give for Lac and Lip some numbers in the text or a figure or mention if they were under the detection limit (and what was that).

Lines 263-268: This sentence is too long and the meaning unclear. Use shorter sentences with clearer explanations especially when it comes to conclusions.

Line 265: Do you mean that the highest MniP production for C/N-58 OMSW is at day 30 and for the C/N-45 OMSW/AS is at day 15? Are there significant differences between MniP production at day 15 and day 30 for C/N-58 OMSW? You should mention if there are significant differences (because this is not clear from figure 2b-they look the same), otherwise reconsider your conclusion.

Line 266-268: “which could explain….figure 1b” rephrase and explain your conclusion. Its not clear what you mean.

Line 268-275: again a very large sentence….

Line 268: Since MnP has a higher redox potential than other enzymes such as laccase. ….something is missing from the sentence?

Line 270: Consider replacing “good” with “high” and “phenols” with “phenolic”.

Line 271: Rephrase (..the secreted quantity could be enough for the recovery of this ...or the secreted amount could be recovered so that this valuable enzyme could be used for….)

Line 272: Since it has

Line 276: isolated from

Line 285: Text is missing at the beginning of the sentence (probably a paragraph).

Line 298: effect on

Line 340: parameters

Line 341: such as MnP and also for

Line 352: consider “to” instead of “that can”

Author Response

Thank you for your comments 

Please see the attachment 

Author Response File: Author Response.pdf

Reviewer 3 Report

The study is interesting, relevant, and of general interest to the readers of this journal containing important information about the use of aquaculture sludge (AS) in olive mill waste fungal treatment (the main factor of novelty). The use of AS aims for a sustainable way to supplement the original substrate concerning C/N ratios. This study is particularly relevant from the circular economy point of view.

The objectives of this work are clearly stated and comprehensively justified.

The introduction covers both old and new references concisely and perfectly integrates the theme's main aspects.

This article is well written, with a good organization of the contents. The whole experimental design, to test the effect of AS use in enzyme activity and C/N ratio, was carefully elaborated and meticulously presented, integrating the main aspects of the scenario under study. However, some concerns were raised regarding the statistical foundation for some important conclusions (effect of the variables under study). These concerns are detailed as specific comments.

About the presentation of the results, we found it suitable, clear, and concisely written ensuring a proper interpretation and understanding.

The cited core references are recent and appropriate to the discussion. The manuscript is very nicely discussed only some typos were identified.

Specific comments:

#1_L233-L238-Figure 1:

#1.1_ Graph (a), “Solid phase (0 day)” the error bars are equal. Please check the original data, we strongly believe that there is some typo in the data.

#1.2_ Graph (b) error bars are lacking.

#2_ L218-245- “Assessment of aquaculture sludge effect”:  

The foundation of experimental research involves testing not only the research hypothesis but also the statistical hypothesis. The second seems to be lacking.

A suitable statistical test (e.g. F test) needs to be performed to conclude if there is a significant difference among the considered groups/treatments. Please provide the results of the analysis of variance, to consubstantiate the conclusions about the effect of those variables, indicating clearly what is the control, which variables are being compared to the control, and the level of significance adopted.

 

The definition of control in the experiment seems not to be clearly defined. Please clarify this aspect.

Author Response

Thank you for your comments 

Please see the attachment 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

We thank the authors´ effort to address and resolve the concerns raised.