Biodegradation of Brown 706 Dye by Bacterial Strain Pseudomonas aeruginosa

Round 1

Reviewer 1 Report

This paper studies biological degradation of a specific dye, say, Brown 706 with various microorganism. Authors found that Pseudomonas aeruginosa  (P. aeruginosa) was most efficient strain for biological degradation, judging from the fading its colors (by UV/Vis spectroscope). For the representative strain, authors found optimal physicochemical conditions for better degradation, by changing working temperature, pH, dye concentration, salt concentration, and glucose concentrations. Authors also attempted to measure or identify the metabolite of Brown706 through GC/MS, FTIR, and NMR, and later concluded that Brown706 mostly yielded p-Xylene as degraded metabolite.

This paper was rather preliminary and well suited for entering biological degradation of chemical pollutants, but has many implication to some extent.

In the first place, I’d like to ask why authors chose Brown706 as a model compound or representative pollutant. Is it really a major component from all environments of the globe?

Second, in the GC spectrum I can’t see any peak in Figure10, but can recognize the mass spectrum. Is it miss printing or are authors omitting data during the course of data preparation?

Third, in favor of authors’ argument, have authors tried to degrade Brown706 with simple mixture of the mentioned enzymes like laccase, azoreductase, peroxidase, and/or other enzymes.

Overall, I recommend publishing this paper after minor revision.

Some typos: line 238 --- which helps increase

Author Response

Reviewer 1

This paper studies biological degradation of a specific dye, say, Brown 706 with various microorganism. Authors found that Pseudomonas aeruginosa  (P. aeruginosa) was most efficient strain for biological degradation, judging from the fading its colors (by UV/Vis spectroscope). For the representative strain, authors found optimal physicochemical conditions for better degradation, by changing working temperature, pH, dye concentration, salt concentration, and glucose concentrations. Authors also attempted to measure or identify the metabolite of Brown706 through GC/MS, FTIR, and NMR, and later concluded that Brown706 mostly yielded p-Xylene as degraded metabolite.

This paper was rather preliminary and well suited for entering biological degradation of chemical pollutants, but has many implication to some extent.

Thank you worthy reviewer, for the encouraging remarks.

In the first place, I’d like to ask why authors chose Brown706 as a model compound or representative pollutant. Is it really a major component from all environments of the globe?

• The selected dye is mostly used for coloring lather throughout the world which in some countries is disposed up as such into environment where water bodies are its ultimate sink. In water bodies a number of microbes including bacteria are living there which help in decolorization. In this study we have tried to find the chemical nature of metabolites which indeed is required. Xylene as biproduct by itself. Is toxic substance. The dye is used widely that is why it has been selected.

Second, in the GC spectrum I can’t see any peak in Figure10, but can recognize the mass spectrum. Is it miss printing or are authors omitting data during the course of data preparation?

• Worthy reviewer, to spare space that have been placed under one figure caption. If we magnify it then every thing is clear. The fist graph is GC chromatogram while the rest are GC-MS chromatograms.

Third, in favor of authors’ argument, have authors tried to degrade Brown706 with simple mixture of the mentioned enzymes like laccase, azoreductase, peroxidase, and/or other enzymes.

• Sorry worthy reviewer, we do not have the facility to isolate these enzymes from bacteria. Also the enzymes are expensive to buy.

Overall, I recommend publishing this paper after minor revision.

• Thank you worthy reviewer.

Some typos: line 238 --- which helps increase

• Corrected accordingly

Reviewer 2 Report

I have reviewed this manuscript carefully. It has several flaws that should be addressed. My specific comments are listed below:

1. Page 1 line 24: Benefits
2. Introduction, First sentence: Do you think the population has grown due to new technologies? I think this sentence is incorrect.
3. “to ensure life of human population”, I think you mean to ensure the healthy lives of the human population.
4. What were the conditions for degradation when all 15 strains were tested? Please mention in the text as well as in the legends of figure 2. We do not know under some other conditions; another strain can give more impressing results. What were the criteria for initial experiments?
5. All the optimization experiments are conducted in triplicate, and plots are shown as mean values and error bars. Please add error bars for all the figures from 3 to 8.
6. Why was the effect of the bacteria concentrations/counts not studied? Even it is not clear at what bacteria concentration were all the experiments performed.
7. GC-MS showed the presence of several organic compounds in the degraded samples. These compounds can be even more toxic than the dye itself. This logic cannot be accepted that these compounds are coming from organic solvents. If this is the case, high purity solvents should have been used. These compounds may affect the dye concentration measurement as well. This issue should be addressed seriously.
8. How can this kind of experiment be implemented at the commercial level?

Author Response

Reviewer 2:

I have reviewed this manuscript carefully. It has several flaws that should be addressed. My specific comments are listed below:

1. Page 1 line 24: Benefits

• Thank you worthy reviewer, it was corrected accordingly.

1. Introduction, First sentence: Do you think the population has grown due to new technologies? I think this sentence is incorrect.

• Thank you worthy reviewer, the sentence was rephrased accordingly.

1. “to ensure life of human population”, I think you mean to ensure the healthy lives of the human population.

• Thank you worthy reviewer for your valuable suggestion.

1. What were the conditions for degradation when all 15 strains were tested? Please mention in the text as well as in the legends of figure 2. We do not know under some other conditions; another strain can give more impressing results. What were the criteria for initial experiments?

• Worthy reviewer, the condition were: 20 ppm concentration, 37ºC, 15 ml broth medial mixed with bacterial inoculum taken from bacterial suspension of 10⁸-10⁹ CFU/ml, incubated for 3 days at neutral pH

1. All the optimization experiments are conducted in triplicate, and plots are shown as mean values and error bars. Please add error bars for all the figures from 3 to 8.

• Error bars were accordingly added to figures pointed out by worthy reviewer.

1. Why was the effect of the bacteria concentrations/counts not studied? Even it is not clear at what bacteria concentration were all the experiments performed.

• The concentration of bacterial inoculum was according mentioned in the revise manuscript. Being living organism it is not possible to accurately determined the bacterial concentration at a given moment as bacterial rapidly undergoes cell divisions that is quite difficult task to monitor, therefore we have not studied the bacterial concentration effect has not evaluated.

1. GC-MS showed the presence of several organic compounds in the degraded samples. These compounds can be even more toxic than the dye itself. This logic cannot be accepted that these compounds are coming from organic solvents. If this is the case, high purity solvents should have been used. These compounds may affect the dye concentration measurement as well. This issue should be addressed seriously.

• Worthy reviewer, our study is about to determined the nature of degraded products. The para xylene by itself is toxic however, our claim is based on the combined results of GC-MS and NMR. We can only made a claim when we have instrumental evidence. At present we have only found evidence for para xylene. Although the other compounds are there in GC-MS graph but we do not have its proof NMR data so we are unable to make such claim. It is general practice that for initial extraction commercial grade solvents are used as they are less expensive (being based in a third world country we cannot afford) and you know it better that also contains a number of impurities. Again our claim should at least be support from NMR which we have only for para xylene.

1. How can this kind of experiment be implemented at the commercial level?

Worthy reviewer, the study aim is to determine the nature of metabolites as there are many reported studies on dyes degradation but there was a dire need to determine the nature metabolites.  In this connection limited studies are there. Such types of study will help in applicabilities of microbe in industries as it ensure the toxic nature of the resultant metabolite. If a very toxic nature metabolite is formed rather dye molecule by itself then decolorization or degradation is of no use. 

Reviewer 3 Report

The manuscript deals with the "bioremediation of dye by Pseudomonas aeruginosa". The main concern is the novelty of the paper. May you please clarify the novelty of your study in the Introduction.

During recent years, the unprecedented development of industrial and urban activities has led to a significant increase in wastewater discharge into the environment, often polluted with harmful organic contaminants (e.g., dyestuffs). Thus, the separation/elimination of these contaminants from water bodies is a goal that must be accomplished to ensure human and environmental safety.

1.  Write keywords alphabetically.

2. Page 1, Line 18; "...due their compatibility... " Should be corrected as "..due to their compatibility..."

3. Page 2, Line 60; "Broadly, these methods are classified as physical, chemical, and biological methods." Try to bold the lack of previous studies? For instance, physicochemical methods produce the secondary pollution.

4. Page 2, Line 69; "Pseudomonas aeruginosa bacteria showed high efficacy for the degradation of the selected dye as compared to other bacterial strains" So what is the novelty of your study?

5. Page 5, Line 201; "3.1 Highly potent bacterial strain" Too short! Please discuss the results and compare them with previous studies.

6. Page 6, Line 210; "3.2. Effect of dye concentration on degradation" Same comment as above.

7. Page 6, Line 220; "3.3. Effect of pH" Same comment as above! Why maximum degradation was recorded in neutral pHs? 

8. Page 7; Line 229; "3.4. Effect of temperature" Same comment as above.

9. Quality of figure 10 should be improved!

10. Quality of figures 13 and 14 should be improved.

Author Response

Reviewer 3

The manuscript deals with the "bioremediation of dye by Pseudomonas aeruginosa". The main concern is the novelty of the paper. May you please clarify the novelty of your study in the Introduction.

• The novelty statement has been added to introduction section accordingly as: Bacteria being a dweller of varied environments and conditions is preferably used for such purposes. Also, its multiplication power and growth are high that is why it has been used previously for the degradation/decolorization of dyes. However, there are limited studies where researchers have studied nature of resulting metabolites of degradation. There is a dire need to determine the nature of metabolites of dyes as sometime the metabolites formed are more toxic than the original dye and in that case such type of transformations are worthy of nothing [1, 2]. Brown 706 is extensively used in coloring of lather, cloth etc has not been subject to such type of studies before

During recent years, the unprecedented development of industrial and urban activities has led to a significant increase in wastewater discharge into the environment, often polluted with harmful organic contaminants (e.g., dyestuffs). Thus, the separation/elimination of these contaminants from water bodies is a goal that must be accomplished to ensure human and environmental safety.

• Thank you worthy reviewer, the statement was accordingly inserted into last paragraph of introduction section.

1. Write keywords alphabetically.

• The worthy reviewer suggestion was endorsed accordingly

2. Page 1, Line 18; "...due their compatibility... " Should be corrected as "..due to their compatibility..."

• Corrected accordingly

3. Page 2, Line 60; "Broadly, these methods are classified as physical, chemical, and biological methods." Try to bold the lack of previous studies? For instance, physicochemical methods produce the secondary pollution.

• Worthy reviewer, thank you very much the required detail was added accordingly into the introduction section.

4. Page 2, Line 69; "Pseudomonas aeruginosabacteria showed high efficacy for the degradation of the selected dye as compared to other bacterial strains" So what is the novelty of your study?

• The novelty statement has inserted into introduction section accordingly. A bacteria has high efficiency of decolorization is just a criteria of selecting efficient organism. The novelty of the study is to determine nature of metabolites formed and proposed a mechanism how they were formed which has not been investigated before. Also, brown 706 has not been subjected to such types of studies before.

5. Page 5, Line 201; "3.1 Highly potent bacterial strain" Too short! Please discuss the results and compare them with previous studies.

• Worthy reviewer, the fact that bacteria are capable of decolorization of dyes is known. Among the bacterial strains some are potent while other are less effective. We have just ensure which is more potent. As stated before such type of study has not been done before therefore we are unable to make such comparisons.

6. Page 6, Line 210; "3.2. Effect of dye concentration on degradation" Same comment as above.

• The required detail was inserted accordingly

7. Page 6, Line 220; "3.3. Effect of pH" Same comment as above! Why maximum degradation was recorded in neutral pHs? 

• The required detail was inserted accordingly

8. Page 7; Line 229; "3.4. Effect of temperature" Same comment as above.

• The required detail was inserted accordingly

9. Quality of figure 10 should be improved!

• The figure quality was improved accordingly

1. Quality of figures 13 and 14 should be improved.

• The figure quality was improved accordingly

Round 2

Reviewer 2 Report

My comments have been addressed.

Reviewer 3 Report

Reviewers' comments have been addressed.