Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Abstract

Consider that the abstract is very small version of the paper. It must contain all the sections: Introduction, materials and methods, results and conclusion. The presented abstract does not accomplish this rule. It is confusing when presenting the germination experiment. Abstract must be rewritten.

In introduction authors must justified why they are using RR-141 dye as a model of study. Is it commonly used in textile industry?

Author Response

Dear, thank you sincerely for your thoughtful insights. We have revised the summary in alignment with your feedback. Additionally, in response to your suggestion, we incorporated an introduction elucidating the rationale behind utilizing the RR 141 dye, emphasizing its prevalence in the textile industry.

Reviewer 2 Report

Comments and Suggestions for Authors

The MS is generally logically organized and well written. In Abstract, I suggest to avoid verbal hyperboles such as "The impact of azo-dyes on the environment can be devastating". 

Specific comments:

Line 46:" In this sense, the activated sludge approach generates  approximately 500 kg of sludge per m3 of wastewater" This seems a very high amount, it probably refers to a specific humidity content. It would be preferable to refer this to dry sludge at a specified level of humidity.

"and does not act on azo dyes" I suggest "is not effective for azo dyes degradation"

Figure 1: since it is stated that "decolorization occurred primarily during 48 h of incubation" it would be interesting to show images at that time. I suggest "within 48 hours" rather than "during"

Table 2. It appears that some text is missing

 

Comments on the Quality of English Language

Minor refinements recommended

Author Response

Dear, thank you for your valuable suggestions. We have revised the abstract accordingly, incorporating a comparative table in section 3.5 to present the decolorization and removal results of the RR 141 dye obtained through various techniques. It is important to note that a constant temperature (30 ºC) was maintained throughout all experiments, with no variations. The variations and their potential impacts of pH on degradation are discussed in section 3.2.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled " Screening of azo-dye degrading bacteria from textile industry wastewater-activated sludge" by Grazielly Maria Didier de Vasconcelos and coworkers, discussed the impact of azo-dyes on the environment can be devastating. Activated sludge is com- 15 posed of aerobic and anaerobic bacteria and fungi dye-degrading (decolorization and mineralization).

These changes are required

1.     Add the values of dye degradation that were measured by different techniques and make it part of the abstract.

2.     A table must be added that gives the dye degradation values using different techniques.

3.     All analysis done related to biodegradation was studied at which temperature

4.      What is the pH during these analyses?  

5.     What are the effects of both pH and temperature on dye degradation?

Author Response

Dear, thank you for your constructive feedback. We have carefully revised the text, implementing changes to address and incorporate your suggestions.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors General Comments:

This manuscript titled “Screening of azo-dye degrading bacteria from textile industry wastewater-activated sludge” presents interesting findings on different bacteria that reside in wastewater-activated sludge and help degrade dyes. I find this manuscript is novel and valuable to the journal’s readership as it discusses valuable kinetics and phytotoxicity aspects in addition to dye degradation potentials. Comparison of dye degradation by isolated strains and in consortia is important.

Even though the abstract, methodology, and title imply screening of dye-degrading bacteria from real textile industry wastewater using the MALDI-TOF-MS technique, I did not see any findings related to this in the main text or in the appendix. Instead, I feel authors have identified species through database search. I recommend getting this clarified before considering this manuscript for publication.

Several specific comments are listed below to improve the manuscript.

 

Specific comments 

Line Number	Comment
15-16	This sentence seems incomplete. Did the authors mean dyes-degrading bacteria and fungi? Suggest rephrasing.
47	The phrase “does not act on azo dye” is not clear to me.
49	“These habitats” do the authors refer to wastewater-activated sludge? suggest specifying
53	“However” doesn’t fit here. Isn’t it a plus point?
54-55	Better to discuss a little more about this technology as it is the novelty of this study and offers several benefits over traditional microbial identification techniques.
54	Define the acronym at its first appearance. E.g.: MALDI-TOF-MS, HPLC, RR-141, etc.
83-84	Suggest reordering phrases
113	Shouldn’t it be “decolorization potential” instead of “potential decolorization”
116	Do these three mediums favor certain species? What’s the rationale behind using three different media?
122-123	Does this culture medium refer to the one detailed in section 2.2? or were distinct bacteria strains used instead of bacteria isolates from real activated sludge?
132	“both” should be changed to all. What are the strains employed?
125-133	How was decolorization measured in culture grown on solid media in plate assays? I am certain UV absorption measurement is not feasible for solid media.
174-196	Section 3.1 and Table 1 are more suitable under methodology to make the methodology clearer.
Table 1	What is given by “score”?
206	Figure 1 only shows 0 hr and 168 hrs. If major changes occur in between, it is better to include a few intermediate images where those rapid changes occur.
Figure 1	Shouldn’t all plates at the starting point be of the same color? What caused the change? Culture media?
214	The first phrase is not needed for the sentence as it doesn’t explain anything shown in the figure. This phrase could be used in line 219 instead.
214-218	Suggested adding references as they are not deduced from results for the current study
226	“Fig” and “figure” are used here and there. Suggest using a consistent style.
231	Even the liquid medium results support this selection not only the solid medium
278	Should be “Azoreductase”
284-285	Repetition of lines 266-268
288	Define abbreviations at their first appearance
296-297	Empty table?
373	What is meant by “item”? “section” would be more appropriate
383-385	The figure caption is not descriptive enough
Figure 5-a	the x-axis title should be corrected to “growth media” rather than “treatment”
387-390	Even though the number of seeds germinated is a little low in RR141 media, germination nature looks better than tap water. I feel the sample size is too small to make solid conclusions here. Was the experiment repeated?

Comments on the Quality of English Language

Minor rephrasing of sentences is suggested for clarification. 

Author Response

Dear, thank you for your valuable suggestions. We have thoroughly reviewed the text and tried to incorporate each of your recommendations.

In selecting culture media for our comparative analysis of species, we opted for commonly used bacterial growth media to avoid selectivity (question about line 116). Additionally, we included a saline medium lacking a carbon source (MS culture medium) to assess the capability of bacteria to use RR 141 dye as a carbon.

Concerning your inquiries about the discoloration analysis on solid media (question about line 125 -133), we visually assessed it by comparing culture media with each other and with a non-inoculated control medium. Any color variations observed in the solid samples at time 0 h (figure 1) may be attributed to ambient lighting during photography, which was not apparent during the visual evaluation. Unfortunately, we do not have photos of intermediate times, but the discoloration process was gradual (question about line 206).

Regarding your question in lines 122-123, the mentioned culture media are BHI, MSG, and MS, specifically in liquid form, with BHI corresponding to the medium detailed in section 2.2. Additionally, all bacteria employed in this investigation were isolated from sludge from a textile industry waste treatment facility.

The values shown in Table 1 assigned as "score" represent the similarity of the species identified with the database used in the identification technique. A score > 2.3 indicates “highly probable identification”, between 2 and 2.299 means “confident identification of the genus, and probable identification of the species”, between 1.7 and 1.999 indicates “probable identification of the genus” and a score < 1.7 indicates “unreliable identification”. We added this description to the article methodology. In addition, please see the following link https://www.bruker.com/en/products-and-solutions/microbiology-and-diagnostics/microbial-identification.html

Considering your query about the phytotoxicity analysis, it's important to note that the germination process is inherently complex. Seeds may not germinate well in water alone because they lack essential minerals and nutrients crucial for expediting germination. Despite this, the results suggest a trend toward dye toxicity. Therefore, a comprehensive evaluation of phytotoxicity involving more samples and tests with different cultivars could be a compelling subject for future research.