Evaluation of a Microbial Consortium and Selection of a Support in an Anaerobic Reactor Directed to the Bio-Treatment of Wastewater of the Textile Industry

Round 1

Reviewer 1 Report

Presented manuscript entitled “Evaluation of a Microbial Consortium and Selection of a Support in an Anaerobic Reactor directed to the Bio-treatment of Wastewater of the Textile Industry” is written in a very chaotic way and needs major corrections before publishing. I assume, Authors directly translated the text to English, while there are several “leftovers”, e.g., “4 g/L de (NH₄)₂SO₄”, “nonifenol” or not very understandable sentences, e.g., “There are several treatment methods, of which the biological being the most feasible”. There is also a lot of words divided in the middle with a dash, e.g., “deter-mined”, “con-ducted” etc., as if an inaccurate copy-paste commend was used.

Below, please find my detailed comments, doubts, and questions.

·         Please use the complete names of used dyes, written with capital letters, as there are they proper names, e.g., “Reactive Red 141”, “Direct Red 23” etc. As for “yellow 84”, there are two completely different dyes marked with this number: Reactive Yellow 84 and Disperse Yellow 84. Please verify, which one was used.

·         The bacteria and fungi species in Abstract should be written in italics.

·         Some parts of the text are marked green.

·         There is almost no mention about work novelty, in comparison to the literature. Moreover, there is lack of references from last 5 years (the newest article is from year 2013).

·         Lines 45-47: “The wastewater is characterized by being alkaline and having high levels of the biochemical oxygen demand (BOD) and the chemical oxygen demand (COD)” – please add more specific data about COD and BOD (values).

·         In the chapter 2.4. there is a lack of subscripts.

·         Chromium(VI) should be written without space.

·         The shortcuts MRS and MRA should be explained when first appear in the text.

·         Chapter 3.2.: “Cell density increased from 105 CFU/ml to 1010 CFU/ml for yellow 84 and red 141 as well as 109 CFU/ml for blue Navy 171, at pH 6.  The population decreased to 108 CFU/ml in red 141 and 107 CFU/ml with both Navy blue 171 and yellow 84” – this sentence is completely incomprehensible. What were the initial values of cell density?

·         The Results section is very poorly described. Please expand it.

Author Response

Response Letter to the expert reviewer

Dear Reviewer,

As authors of the manuscript “Evaluation of a Microbial Consortium and Selection of a Sup-port in an Anaerobic Reactor directed to the Bio-treatment of Wastewater of the Textile Industry”, we appreciated a lot your suggestions and comments on the document, as we are certain and convinced, that they have been useful to enrich the fluency and clarity of the entire article. We also confirm that the writing in English has been thoroughly reviewed and accordingly improved.

Below, we will detail the changes made and you will be able to find them all exposed and answered since the responses to each suggestion and comments are given in bold.

Review #1:

There is also a lot of words divided in the middle with a dash, e.g., “deter-mined”, “con-ducted” etc., as if an inaccurate copy-paste commend was used.

Existing hyphens were removed throughout the document.

Please use the complete names of used dyes, written with capital letters, as there are they proper names, e.g., “Reactive Red 141”, “Direct Red 23” etc. As for “yellow 84”, there are two completely different dyes marked with this number: Reactive Yellow 84 and Disperse Yellow 84. Please verify, which one was used.

In the investigation, reactive dyes were used that are specified between lines 103 -109 and throughout the document, the indicated capital letters were revised.

The bacteria and fungi species in Abstract should be written in italics.

Changes have been made based on your assertive suggestion

Some parts of the text are marked green.

The green colour was removed from the indicated words.

There is almost no mention about work novelty, in comparison to the literature. Moreover, there is lack of references from last 5 years (the newest article is from year 2013).

Citations were updated based on your recommendations

Lines 45-47: “The wastewater is characterized by being alkaline and having high levels of the biochemical oxygen demand (BOD) and the chemical oxygen demand (COD)” – please add more specific data about COD and BOD (values).

Following their suggestions, more specific data was placed, which are located between lines 45-50

In the chapter 2.4. there is a lack of subscripts.

The numbers have been placed in subscript format

Chromium(VI) should be written without space.

Based on your kind suggestions, the space has been removed throughout the document.

The shortcuts MRS and MRA should be explained when first appear in the text.

In line 153, the meaning of the acronyms has been included.

Chapter 3.2.: “Cell density increased from 105 CFU/ml to 1010 CFU/ml for yellow 84 and red 141 as well as 109 CFU/ml for blue Navy 171, at pH 6.  The population decreased to 108 CFU/ml in red 141 and 107 CFU/ml with both Navy blue 171 and yellow 84” – this sentence is completely incomprehensible. What were the initial values of cell density?

From Line 206 it has been explained what the initial value of the cell density was.

The entire document was reviewed.

Once again and with all due respect, we are very thankful for your comments and corrections, which helped to see a few unclear parts and or even faults of our side within our manuscript. With your comments we were able to smooth the text, clarify missing parts or wrong spellings, which resulted to a much better than the initial version of this current study. Thanks a lot on behalf of all authors

Reviewer 2 Report

Text editing:

- Correct hyphenation throughout the text (e.g. line 14: "un-fixed", line 15: "environ-mental", line 43: "de-composition", line 48: "sol-vents", line 50: "be-tween” etc.

- Unify throughout the text in Latin names. They should be written in italics. E.g. line 21, 28-30, etc.

- Line 33: There is a incorrectly written a number. Instead of a decimal point, there is a comma and a space.

- Line 55: 'nonifenol' - if it is a detergent, it says nonylphenol.

- Line 87: I am sure that wastewater is written together rather than 'waste water'

- Check and correct superscripts and subscripts throughout the text, eg line 105: "3x108 CFU / mL), as well as line 120:" K2CrO4", etc.

- Line: 157-158: "bottle plastic" - is the plastic from which the bottles are made? Here it could also be specified which plastic it is. It is mostly PET - polyethylene terephthalate.

- Line 445: “chromium IV…” replace VI.

Questions and comments on the scientific part:

- Line 93-94: What was exposed to the air? According to the literature, it was activated sludge from the treatment plant. However, this is not entirely clear from this sentence. Fill in.

- Line 107: Insert the composition of the tested azo dyes Navy blue 171, red 141 and yellow 84, preferably chemical formulas.

- Line 186: How did the I5-ESPE and I6-ESPE consortium differ that the results were so different?

- Line 187 and more: p = 0.00 - is it correct that it is zero? Either write what is the real number, e.g. 0.0000001 or write <0.00. If the statistical software plots a p value of 0.00, it means that the value is very low, with many "0" before any other digit. For example, in SPSS, you can double-click it to see the current value.

- Line 255-257: Figure 11. What exactly does the graph represent? Representation of which species or is it a change in the representation of one specie before and after treatment?

- Row 266-269: What was the fraction size used for each material?

- Tab. 2 and lines 396-398: Can the dye removal not be affected by sorption on the material used (coconut shell)? Has the adsorption capacity been tested?

Author Response

Response Letter to the expert reviewer

Dear Reviewer,

As authors of the manuscript “Evaluation of a Microbial Consortium and Selection of a Sup-port in an Anaerobic Reactor directed to the Bio-treatment of Wastewater of the Textile Industry”, we appreciated a lot your suggestions and comments on the document, as we are certain and convinced, that they have been useful to enrich the fluency and clarity of the entire article. We also confirm that the writing in English has been thoroughly reviewed and accordingly improved.

Below, we will detail the changes made and you will be able to find them all exposed and answered since the responses to each suggestion and comments are given in bold.

Review #2

Correct hyphenation throughout the text (e.g. line 14: "un-fixed", line 15: "environ-mental", line 43: "de-composition", line 48: "sol-vents", line 50: "be-tween” etc.

The hyphens in the text were carefully revised and removed.

Unify throughout the text in Latin names. They should be written in italics. E.g. line 21, 28-30, etc.

Throughout the text, the Latin names were corrected and placed in italics.

- Line 33: There is a incorrectly written a number. Instead of a decimal point, there is a comma and a space.

The aforementioned bug was fixed

Line 55: 'nonifenol' - if it is a detergent, it says nonylphenol.

The aforementioned bug was fixed

Line 87: I am sure that wastewater is written together rather than 'waste water'

Separate words were joined.

Check and correct superscripts and subscripts throughout the text, eg line 105: "3x108 CFU / mL), as well as line 120:" K2CrO4", etc.

Throughout the document you can see that we have corrected the superscripts and subscripts

Line: 157-158: "bottle plastic" - is the plastic from which the bottles are made? Here it could also be specified which plastic it is. It is mostly PET - polyethylene terephthalate.

In lines 161-162, the material that is made of “PET” plastic bottles was included.

Line 445: “chromium IV…” replace VI.

In 451 you can verify the change made

Line 93-94: What was exposed to the air? According to the literature, it was activated sludge from the treatment plant. However, this is not entirely clear from this sentence. Fill in.

In lines 98-99, it is indicated that, by exposure to air, the microbial consortia were obtained

Line 107: Insert the composition of the tested azo dyes Navy blue 171, red 141 and yellow 84, preferably chemical formulas.

On lines 112-113, the corresponding formulas were written

- Line 186: How did the I5-ESPE and I6-ESPE consortium differ that the results were so different?

In lines 201-203, Figure 2, it is specified that there have been significant differences I5-ESPE and I6-ESPE in color reduction for each inoculum

Line 187 and more: p = 0.00 - is it correct that it is zero? Either write what is the real number, e.g. 0.0000001 or write <0.00. If the statistical software plots a p value of 0.00, it means that the value is very low, with many "0" before any other digit. For example, in SPSS, you can double-click it to see the current value.

Following your suggestions on lines: 192 and 203, <0.00 was written

Line 255-257: Figure 11. What exactly does the graph represent? Representation of which species or is it a change in the representation of one specie before and after treatment?

Between lines 255-260, it is specified in detail what the graph represents

Row 266-269: What was the fraction size used for each material?

Between lines 158 -168, the parameters for which the materials were selected are indicated: porosity, density and specific weight

Tab. 2 and lines 396-398: Can the dye removal not be affected by sorption on the material used (coconut shell)? Has the adsorption capacity been tested?

In this investigation, the absorption capacity of the coconut shell was not verified.

The entire document was reviewed.

Once again and with all due respect, we are very thankful for your comments and corrections, which helped to see a few unclear parts and or even faults of our side within our manuscript. With your comments we were able to smooth the text, clarify missing parts or wrong spellings, which resulted to a much better than the initial version of this current study. Thanks a lot on behalf of all authors

Reviewer 3 Report

Brief Summary

The paper presents efforts to address the problem of wastewater from the textile industry by developing and testing microbial consortia capable of removing COD and other contaminants. The authors develop two candidate consortia and identify one as being more effective. They test three potential substrates for retention of microbial community in an anaerobic reactor, identifying coconut shell as demonstrating highest potential suitability of the three. The paper presents clear figures demonstrating differences between treatments and a good discussion of the possible reasons for higher efficacy of coconut shell.

The aims of the paper do not mention testing of appropriate support media, which appears to be one of the main aims, separate to development of the microbial consortia. The hypotheses tested in the paper are not clearly stated. It is not clear if there was any replication, and for microbial community analysis there really should be.

It is not clear if the wastewater was sitting in the reactor for the duration or flowing through, as no flow rates are mentioned. Flow/loading rates and retention time are core concepts in wastewater treatment, without which the data may not be relevant.

It is not clear that the COD and contaminant reductions are microbially mediated, it may well be that the coconut shell absorbed the contaminants and over time would become saturated?

The introduction mentions the desired characteristics of suitable support media, one of which is "porosity" - it is therefore not clear why plastic would be chosen, given its low porosity. Explanation in Line 382-385 is inadequate, needs to be expanded with reference to previous work.

Lines 201-205 are not clear. Did cell density increase from 105 to 109 CFU/ml for Navy 171? What was +/- of that? Was it significant? 

Line 202-203 - The population decreased, from what?

Consistently hyphenated random words throughout, eg Line 210, yel-low; Line 233, consorti-um; Line 267, bio-mass

Line 213 - What does "the initial cell density for phenol removal.. increased" mean? Not clear

Line 219-223 - reword, unclear

Line 234-235 - Are you sure that zinc was microbially removed?

For all figures - are there replicates or is n=1? Error bars would help depict significance

Line 251 and 337 - Typo - Sthapylococcus

Figure 11 - not very clear

Table 2 - is COD removal rate higher or lower in plastic vs coconut? Table 2 lists a higher number, but is this the concentration in the effluent? Discussion in Line 396-398 indicates coconut is better?

Line 284-286 - if the cruciality of co-substrate in anaerobic conditions is an established fact (as stated) then why do a study without including co-substrate?

Line 318 and 321 - typo - Echerichia

Author Response

Response Letter to the expert reviewer

Dear Reviewer,

As authors of the manuscript “Evaluation of a Microbial Consortium and Selection of a Sup-port in an Anaerobic Reactor directed to the Bio-treatment of Wastewater of the Textile Industry”, we appreciated a lot your suggestions and comments on the document, as we are certain and convinced, that they have been useful to enrich the fluency and clarity of the entire article. We also confirm that the writing in English has been thoroughly reviewed and accordingly improved.

Below, we will detail the changes made and you will be able to find them all exposed and answered since the responses to each suggestion and comments are given in bold.

Review#3

The aims of the paper do not mention testing of appropriate support media, which appears to be one of the main aims, separate to development of the microbial consortia. The hypotheses tested in the paper are not clearly stated. It is not clear if there was any replication, and for microbial community analysis there really should be.

According to the problem mentioned in the abstract, the objective of this work is:

Evaluate a microbial consortium obtained from wastewater from the textile industry to be used as biomass in an anaerobic biological treatment reactor with a new support medium, in this case, the supports evaluated are rock, plastic and coconut shell.

The bacterial inocula that showed growth under anaerobic conditions in the modified MP medium enriched with 100 mg/L of reactive textile dyes Navy Blue 171, Red 141 and Yellow 84 separately were compared to select the best remover inoculum. The pH of the culture medium was 6. In each tube with dye, 0.1 mL of the mother bacterial culture was seeded, with a cell density of about 3 x 108 CFU/mL determined by counting in deep seeding. From each inoculum, three repetitions were made for each colorant. The variable evaluated was the percentage of color reduction calculated by the relationship established between the spectrophotometric reading before and after the bacterial action.

The selection of the inoculum was made by measuring the color reduction by spectrophotometry and organizing the data obtained within a Completely Randomized Experimental Design (DCA), in which each inoculum constituted a different treatment. Three repetitions were made of each treatment. A DCA was applied to each color and the variable evaluated in all cases was the percentage of color removal. It was analyzed if there was a significant difference between the percentages of removal of the applied treatments through the ANOVA of one factor. Tukey's test was used to observe how the data obtained were located in groups of homogeneity and thus select the inoculum with the highest capacity to remove color.

It is not clear if the wastewater was sitting in the reactor for the duration or flowing through, as no flow rates are mentioned. Flow/loading rates and retention time are core concepts in wastewater treatment, without which the data may not be relevant.

The inoculum and the synthetic water were placed in a 2:1 ratio and samples were taken every 24 hours to control the adaptation of the biomass inside the reactor, through the analysis of COD, TSS and SSVL, in addition, it was controlled that the pH was keep between 6.5 and 7.6 and a temperature of 35°C. The reactors were operated in semi-continuous flow, extracting 300 mL of treated water and placing 300 mL of synthetic water every 24 hours, maintaining a constant volume of 4 L.

It is not clear that the COD and contaminant reductions are microbially mediated, it may well be that the coconut shell absorbed the contaminants and over time would become saturated?

The initial tests indicated that the microbial consortium I5-ESPE has the capacity to remove contaminants, while later it was tested in an anaerobic reactor to demonstrate that it is feasible to apply the consortium together with the support whose values yielding that there is removal of contaminants and COD. Within the characterization of the support, the porosity, density and specific weight were evaluated, however, the absorption capacity of the coconut shell was not determined. Possibly the composition of the chosen support, that is, the coconut shell influences the absorption of contaminants since there are current studies that mention this support as an absorbent medium for the elimination of heavy metals and its high water retention capacity.

 In this experiment the absorption capacity of the coconut shell is not evaluated. 

which is "porosity" - it is therefore not clear why plastic would be chosen, given its low porosity. Explanation in Line 382-385 is inadequate, needs to be expanded with reference to previous work.

Between lines 390 and 393, the explanation of why to use plastic was written

Lines 201-205 are not clear. Did cell density increase from 105 to 109 CFU/ml for Navy 171? What was +/- of that? Was it significant? 

105 to 109 , better written 10⁵ to 10⁹ as these are exponential values

Line 202-203 - The population decreased, from what?

In line 208, the corresponding clarification was written.

Consistently hyphenated random words throughout, eg Line 210, yel-low; Line 233, consorti-um; Line 267, bio-mass

Throughout the document, a thorough revision of the scripts was carried out.

Line 213 - What does "the initial cell density for phenol removal.. increased" mean? Not clear

Between the 219-220 lines, it was explained better.

Line 219-223 - reword, nuclear

Between lines 225-229 the writing was improved

Line 234-235 - Are you sure that zinc was microbially removed?

Figures 8 and 9 show the values obtained.

For all figures - are there replicates or is n=1? Error bars would help depict significance

All figures are n=1

Line 251 and 337 - Typo – Sthapylococcus

In the document it was corrected

Figure 11 - not very clear

Figure 11 quality improved

Table 2 - is COD removal rate higher or lower in plastic vs coconut? Table 2 lists a higher number, but is this the concentration in the effluent? Discussion in Line 396-398 indicates coconut is better?

Table taken from the original document:

Table 2.- Evaluation of support media with the I5-ESPE microbial consortium in the treatment of textile wastewater.

Among the supports commonly used are rock and plastic. Rock in sizes smaller than 3 cm has been able to generate efficiency losses due to clogging, while plastic allows the adherence of anaerobic microorganisms since methanogenic populations are able to associate with stable solid materials for form beds [98] and it has been proven that plastic as a support medium has achieved a COD removal efficiency of between 70 and 90% [99,100]. An alternative material as a support medium in anaerobic filter is coconut shell due to its large specific surface for adherence of microorganisms, high percentage of voids facilitators of flow (83%), low specific weight with less complex containment infrastructures and long useful life [18]. In our study, the porosity obtained for coconut shell (83.06%) has been higher than for rock (44.24%) and plastic (13.96%). The distribution of the pores and their size guarantees a contact surface for the generation of the biofilm and transfer of the contaminant mass, favoring the microbial adhesion conditions from the initial stages of the process. The specific weight of the supports determines the construction costs, so if the support material is lighter, no external structural effort will be required to sustain the filter medium and the transportation and installation of the material will be lower

Comment: The objective of this part of the text is to propose the coconut shell as an alternative for support in a reactor for treatment of wastewater from the textile industry and is specifically focused on the removal of dyes, which in this case is more efficient compared to plastic. and to the common rock. As in the removal of dyes, COD is better reduced in relation to plastic and common rock.

Line 284-286 - if the cruciality of co-substrate in anaerobic conditions is an established fact (as stated) then why do a study without including co-substrate?

Being a test that was carried out at the laboratory level at a small volume in order to compare and select the support medium that achieved the greatest removal of dye and COD, the use of co-substrate was not taken into account, which is mentioned in the document, however, if the objective is to increase the removal values, the use of co-substrate is necessary

Line 318 and 321 - typo – Echerichia

The entire document was reviewed.

Once again and with all due respect, we are very thankful for your comments and corrections, which helped to see a few unclear parts and or even faults of our side within our manuscript. With your comments we were able to smooth the text, clarify missing parts or wrong spellings, which resulted to a much better than the initial version of this current study. Thanks a lot on behalf of all authors

Round 2

Reviewer 1 Report

Authors answered my questions and corrected the manuscript. In this regard, I suggest acceptance of presented manuscript in present form. 

Reviewer 3 Report

Significantly improved, well done.

Line 404 has a space or / missing between anaerobic and aerobic.