Multi-Objective Statistical Optimization of Pectinolytic Enzymes Production by an Aspergillus sp. on Dehydrated Coffee Residues in Solid-State Fermentation

Round 1

Reviewer 1 Report

Dear Authors,

I would like to acknowledge the importance of the issue described in your paper is rather innovative and SSF method is suitable for Aspergillus fungus. I have some minor suggestions that should be implemented in the manuscript:

• line 86 - please describe methodology from reference 29 (general facts);
• lines 101-103 - please describe what methods are hiden under standard methods numbers;
• line 142 - please describe the abbreviation with the first time when it occurs in the text;
• Table 2 and 3 - I would recommend to put in in the Supplementary Materials;
• line 280 - please describe the unit - gws and #Sp ?
• Figure 2 - improve the quality of the figure;
• Please improve the dsicussion secion, there is little information on comparison of your own results with others' authors researches
• Conclusions (lines 352 -357) - the majority of this section should be removed to introduction, please summarize your own achievements described in this paper

Author Response

Dear Reviewer 1,

Thank you very much for the comments and suggestions made to my manuscript. The changes made, according to your suggestions, appear in the document, underlined in yellow.

Next, I proceed to answer each of your remarks.

• line 86 - please describe methodology from reference 29 (general facts);

According to your suggestion, the following was added (lines 86-89):

A brief description, every two weeks, colonies of Aspergillus sp. were seeded in a solid sporulation medium containing (per litre): cassava flour (40 g), KH₂PO₄ (2 g), (NH₄)₂SO₄ (1 g), CaCl₂ (1 g), CO(NH₂)₂ (1 g), and agar (15 g). The pH was adjusted to 5.6.

• lines 101-103 - please describe what methods are hiden under standard methods numbers;

The following paragraph was added (see lines 104-109):

All analyzes were performed according to well established and accepted methods. Reducing sugars (according to AOAC Method 985.29-1986(2003): Total dietary fiber in foods. Enzymatic-gravimetric method), pH (AOAC method 22.061-1980: Glass Electrode Method - Official Final Action), ash (AOAC method 925.51-2002: Ash in Canned Vegetables), and moisture and dry matter (ISO 6540:2021: Maize - Determination of moisture content (on milled grains and on whole grains)) were determined.

• line 142 - please describe the abbreviation with the first time when it occurs in the text;

… relative humidity (RH)… (see line 148)

• Table 2 and 3 - I would recommend to put in in the Supplementary Materials;

As you suggested, I should put tables 2 and 3 into "supplementary materials". However, as the work is not too extensive, I believe that tables 2 and 3 could explain the design of experiments carried out and the quality of the quadratic models obtained.

• line 280 - please describe the unit - gws and #Sp ?

…. g of wet substrate (gws)…. (see line 119)

…. number of spores (#sp)… (see lines 126-127)

• Figure 2 - improve the quality of the figure;

Figure 2 was enhanced and enlarged…

• Please improve the discussion section, there is little information on comparison of your own results with others' authors researches

To improve the discussion, the following paragraph was added (lines 355-360):

Aeration seems to be related to the increased growth rate of the obligate aerobic Aspergillus sp. It appears to be associated with the production of pectinolytic enzymes, which, as the results suggest, is associated (completed or partially) with the Aspergillus sp. growth. In future experiments, the possibility of controlling the flow of air supplied to the system will be implemented in the installation to establish the relationship between the speed of aeration and the production of pectinolytic enzymes.

• Conclusions (lines 352 -357) - the majority of this section should be removed to introduction, please summarize your own achievements described in this paper

The conclusions were rewritten following the reviewer's recommendations (lines 362-374):

In Ecuador, between 1,600-3,200 tons would generate husk and pulp wet coffee residues. Valuing these wastes would have a meaningful, beneficial impact on the agroecosystems and generate income for the Ecuadorian economy. However, fresh coffee residues have high humidity (>80%), favouring their rapid microbial degradation and limiting their storage and use as a starting raw material for obtaining other products.

The study shown here demonstrates, in the first place, the advantages of dehydrating coffee residues, in the first place, which would allow their storage and use for more extended periods. And secondly, the usefulness of coffee DH-residues to produce pectinolytic enzymes was demonstrated using a local strain of Aspergillus sp. in solid-state culture in the simplest existing SSF bioreactor, a tray-type bioreactor, where relative humidity and temperature were controlled.

Statistical optimization and proper design of response surface experiments made it possible to find the best conditions to produce these enzymes in SSF tray-type bioreactors.

Reviewer 2 Report

The current study reported statistical optimization for pectinolytic enzymes production by Aspergillus sp. on dehydrated coffee residues in solid-state fermentation. Although the results may provide some useful information to the audience working in the similar area, some concerns should be addressed before further consideration.

1. 2.1: Can the Aspergillus sp. being accessible for the audience? Has it been deposited in a public stain bank?
2. line 103: What about the analysis for anti-nutritional ingredients? Say, phytic acid and caffeine from the waste, which may slow down the growth of M.O.
3. line 111: Why a 10^3 spores/mL of inoculum was used since we usualy go above 10^5 spores/mL for solid state fermentation.
4. Table 2: The value of lack of fit went above 0.05 which is good for the EA model. What about the spore count model, which was missing in the text.
5. Some of the parameters lacked the effect of interaction. Can the authors explain why?
6. Table 5: Seems the results from the current study was lower than those from the C28B25 even though the similar materials were used. Can the authors explain why?
7. A more detial discussion was expected. For example, the authors should discuss more about the interaction among/between the parameters; the relationship betwwen the number of spores and the enzyme activity; and the comparison with other publications.

Author Response

Dear Reviewer 2,

Thank you very much for the comments and suggestions made to my manuscript. The changes made, according to your suggestions, appear in the document, underlined in green.

Next, I proceed to answer each of your remarks.

 

• 2.1: Can the Aspergillus sp. being accessible for the audience? Has it been deposited in a public stain bank?

Unfortunately, the strain of Aspergillus sp. isolated in our laboratories is not yet available in accredited strain collections of bacteria and fungi. In collaboration with other universities, we are carrying out molecular and phylogenetic tests to characterize our strain, after which we will be able to deposit it in some of the existing collections, and it will be available to the rest of the researchers.

On lines 90-91, was added:

In collaboration with researchers from other universities, ongoing studies are in process for the phylogenetic characterization of the isolated strain.

 

• line 103: What about the analysis for anti-nutritional ingredients? Say, phytic acid and caffeine from the waste, which may slow down the growth of M.O.

It would have been interesting to know the concentration of caffeine or phytic acid and the presence of other organic substances such as tannins, which could be harmful to ecosystems and whose influence on the growth of Aspergillus sp. and the production and secretion of pectinolytic enzymes by Aspergillus sp. could be elucidated. However, these analyzes have not been performed so far. Although in future investigations, indeed, this recommendation will be accepted.

 

• line 111: Why a 10^3 spores/mL of inoculum was used since we usually go above 10^5 spores/mL for solid-state fermentation.

The content of a single plate was seeded where a one or more gram of difference in the weight of the Petri dish was reached, between the initial and the final seeding, which was usually reached after more than one week of incubation (the time oscillated around ten days). With this procedure, initial values of spore concentration between 1-2 ·10³ spores/mL in liquid inoculum were obtained (the average value was 1.3 ·10³). If instead of one, several Petri dishes had been taken, it would have been possible to have an inoculum close to 10⁵ spores/mL, which would shorten the culture times but increase the risks of contamination by having to increase handling processes.

 

• Table 2: The value of lack of fit went above 0.05 which is good for the EA model. What about the spore count model, which was missing in the text.

The lack of fit can occur if not all the essential terms of the model are included, such as the terms associated with the interactions or quadratic terms. When a quadratic model is chosen, as was the case for the spore count, and all the possible terms that this quadratic model suggests as possible are included, as is the case, the term "lack-of-fit" disappears. However, it is observed that in this model, there is a term (the term AB) whose coefficient in the model is not significant. In these cases, this term in the model could be removed. In this case, it was not done, as it would affect the hierarchy of the model, in the case that takes a decision to exclude the AB interaction coefficient if there had been a non-significant "lack-of-fit" with a p-value of 0.4755 and a better R²-adjusted value of 0.9921 (that of the hierarchical model where the AB interaction coefficient is included is 0.9919) but the model, without the AB term coefficient, would lose its hierarchy.

In the case of the transformed EA quadratic model, all the possible terms of the quadratic model were initially included, and subsequently, those non-significant coefficients that did not compromise the hierarchy of the model were removed.

When a polynomial model includes higher-order terms (such as A²B, AB², and/or A²B²) and excludes lower terms (such as A, B, A², B², or AB) contained in higher-order terms, the model is said to lose its hierarchy; and it is believed that the hierarchy principle is a good practice when fitting experimental values to a polynomial.

In the case presented in Table 2, both quadratic models of the transformed responses include the term AB, despite being - in both cases, a non-significant term, and therefore, both quadratic models are hierarchical.

 

• Some of the parameters lacked the effect of interaction. Can the authors explain why?

For both models, initially, all the terms that the quadratic model allowed (A, B, AB, A², B², A²B, AB², and A²B²) were included.

In the case of the model of the transformed response of S (#sp/gws), it was observed that all the terms were significant (p-value < 0.05), except the term AB, and it was decided to keep it in the model to respect the top of the hierarchy.

For the model of the transformed response of the EA (IU/gws), the procedure was the same initially. All the possible terms of the quadratic model were included. Later, the upper terms whose p-values>0.05 were removed from the model and that with their elimination, the R²-adjusted value of the model increased. In this way, only the terms that were significant (p-value < 0.05) remained in the model. The exception was, as in the case of the previous model, the term AB, which despite being non-significant, must be included so as not to violate the principle of hierarchy. From the initial model of the EA transformed response, for this reason, the term A²B² was removed.

 

• Table 5: Seems the results from the current study was lower than those from the C28B25 even though the similar materials were used. Can the authors explain why?

Indeed, the results of the experiments carried out with a wild strain of Aspergillus sp. shown in our study were lower than those presented by Antier et al. (1993). It is due to a double effect, that of the random mutagenesis by radiation of the fungus genome, which made it possible to select hyper-producing strains of pectinolytic enzymes, and a second effect, related to the presence of oxygen.

In the first case, the radiation may activate redundant genes in the fungus to produce pectinolytic enzymes that are usually recessive or silent or inactivate some proteins, which act as repressors of the synthesis of these own enzymes. Random mutagenesis using radiation can only give good results after long and tedious screening processes and selecting hyper-producing strains.

Through the aeration of the culture of the obligate aerobic Aspergillus sp., the presence of oxygen can promote the removal of CO₂ and better conditions for the growth of Aspergillus sp. As the experiments suggest, the pectinolytic enzymes seem to be products associated (partially or totally) with the growth of the fungus. Thus, as the specific growth rate of the fungus increases with aeration, the production of pectinolytic enzymes must also increase.

 

• A more detail discussion was expected. For example, the authors should discuss more about the interaction among/between the parameters; the relationship between the number of spores and the enzyme activity; and the comparison with other publications.

Reformed part of the discussion (lines 353-355) and added a new paragraph (lines 356-361).

lines 353-355:

… or in another case, superior results were achieved after randomizing mutagenesis and extensive and tedious selection processes with the purpose of finding pectinase-hyperproducing strains [42].

lines 356-361:

Aeration seems to be related to the increased growth rate of the obligate aerobic Aspergillus sp. It appears to be associated with the production of pectinolytic enzymes, which, as the results suggest, is associated (completed or partially) with the Aspergillus sp. growth. In future experiments, the possibility of controlling the flow of air supplied to the system will be implemented in the installation to establish the relationship between the speed of aeration and the production of pectinolytic enzymes.

The "Conclusions" section was completely rewritten (lines 363-375):

In Ecuador, between 1,600-3,200 tons would generate husk and pulp wet coffee residues. Valuing these wastes would have a meaningful, beneficial impact on the agroecosystems and generate income for the Ecuadorian economy. However, fresh coffee residues have high humidity (>80%), favouring their rapid microbial degradation and limiting their storage and use as a starting raw material for obtaining other products.

The study shown here demonstrates, in the first place, the advantages of dehydrating coffee residues, in the first place, which would allow their storage and use for more extended periods. And secondly, the usefulness of coffee DH-residues to produce pectinolytic enzymes was demonstrated using a local strain of Aspergillus sp. in solid-state culture in the simplest existing SSF bioreactor, a tray-type bioreactor, where relative humidity and temperature were controlled.

Statistical optimization and proper design of response surface experiments made it possible to find the best conditions to produce these enzymes in SSF tray-type bioreactors.

Round 2

Reviewer 1 Report

Dear Authors I would like to thank you for all your corrections. I wish you a lot of success in developing your research ideas.

Reviewer 2 Report

I am happy with the revision.