Decolourisation of a Mixture of Dyes from Different Classes Using a Bioreactor with Immobilised Pleurotus ostreatus Mycelium
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript deals with a closely related topic of dye degradation and mentions a significant processing operation; however, minor comments were needed to improve the quality of the paper:
-Authors should mention the reproducibility of each experiment; no statistical test was included?
-Authors should discuss whether the strain conducts sorption of biodegradation (biochemical transformation) of the dye.
-A future perspective could be added in the conclusion.
Author Response
Comments 1: Authors should mention the reproducibility of each experiment; no statistical test was included?
Response 1: The studies were conducted using two parallel bioreactors for each modification in order to limit the possibility of the influence of factors such as temperature fluctuations, differences in the concentration of biomass introduced into the reactor, exposure to light, etc. This information is included in lines 118-122. The graph shows average values for measurements from two reactors, but in most cases the differences in dye concentrations in the reactor were no greater than 15%. Toxicity tests were performed for each reactor of a given modification separately. This information is also added in lines 135-136.
Comments 2: Authors should discuss whether the strain conducts sorption of biodegradation (biochemical transformation) of the dye.
Response 2: Much information has been added to the manuscript and additional graphs have been added which further emphasize the phenomena of sorption and desorption, especially in the case of the Congo Red dye. Lines: 214-220
Comments 3: A future perspective could be added in the conclusion.
Response 3: Thank you very much for this comment and tip. I have added a few sentences regarding further research directions and potential use of one of the strains: The obtained research results are extremely promising, indicating the possibility of using a similar system during the initial treatment of wastewater containing dyes, but focusing only on the BWPH strain under periodic aeration conditions. This type of research should be continued and focused, among others, on the optimization of the process, especially in terms of reducing the toxic impact on the aquatic ecosystem.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis paper is interesting, but a bit short
First, authors need newer references regarding the "Decolourisation of Dyes"
Some organisms mentioned are not in italic (line 81 for example)
Line 87. What previous tests? Where are those results? Please elaborate or provide data
Line 95. BagMixer was introduced - possible grammar mistake?
Line 102. 1 L of air in what time? why 1 L? Did you measure O2 concentration?
Why periodical aeration? It is not clear from your methods
Did you measure biomass concentration? If not why not? If yes, provide the results. It is important to show the biomass concentration change in such environments
Did you measure concentration of glucose during your experiments? How does the concentration of dyes influence the carbon source metabolism?
Why only 10 g/L? Did you do these experiments with higher concentrations? Can initial glucose concentration influence the adsorption or removal of your dyes?
Line 132. Please show those results
Can you prove your claim regarding desorption of dyes? (Line 151)
Line 168. What is strong biomass? This claim is based on what? Or what kind of observations
Line 213. Your tested dyes or products of their degradation are toxic, so my question is: What is the purpose of your research? Decolorization occurred but it is not safe for the environment do discharge this mixture in nature?
Can you show results with individual dyes so one can compare those results to ones with your mixture
Does the mass ratio of dyes influence the degradation or adsorption?
Why didn’t you start with lower concentrations and adapt your biomass to such conditions? And then increase the concentration during time?
Line 219. Names in italic
Line 254. What mechanisms? Explain please
Author Response
Comments 1: This paper is interesting, but a bit short
Response 1: Thank you for this comment. I tried not to over-expand the content unnecessarily. In the current version, in accordance with Reviewer 1's suggestion, I added additional information to the summary.
Comments 2: First, authors need newer references regarding the "Decolourisation of Dyes"
Response 2: I added some new items: 16, 17, 18, 21, 26
Comments 3: Some organisms mentioned are not in italic (line 81 for example)
Response 13: Thank you. Done
Comments 4: Line 87. What previous tests? Where are those results? Please elaborate or provide data
Response 4: Thank you very much for this comment and sorry for the oversight. The citation regarding the immobilization of the mycelium of the BWPH strain and also other fungi has been added (17 and 18).
Comments 5: Line 95. BagMixer was introduced - possible grammar mistake?
Response 5: Thank you very much. BagMixer was used as the device for homogenizing mycelium. I have given this information in brackets in the current version to make it more precise.
Comments 6: Line 102. 1 L of air in what time? why 1 L? Did you measure O2 concentration?
Response 6: To maintain the sterility of the process, air was slowly introduced with a 500 ml catheter syringe through a filter with pores of 0.45 micrometers. The time of air supply was not measured, but the process was carried out gently to thoroughly aerate the reactor. The oxygen concentration in the reactors was not measured because they worked in parallel, the samples taken for analysis were small and maintaining sterility would be difficult. I added information to the manuscript: Air was supplied gently by syringe through a probe equipped with a syringe filter to reduce the possibility of contamination of the bioreactor
Comments 7: Why periodical aeration? It is not clear from your methods
Response 7: I did not add it in the text but added item 21. But the real reason was to find savings if constant aeration is not necessary.
Comments 8: Did you measure biomass concentration? If not why not? If yes, provide the results. It is important to show the biomass concentration change in such environments
Response 8: Biomass was not measured because it is difficult to separate biomass from the carrier - biomass strongly develops inside the carrier, hence no methods known to me are able to extract it from between the carrier structure. No specific amount/mass of carrier was added to the bioreactor, hence it would be difficult to estimate what it is. I tried to do it as accurately as possible, but with such a volume of the reactor the error would be too large. At present, no methods known to me are able to fully estimate the biomass developing in this way. Furthermore, observations show that additionally biomass develops on probes introducing air into the bioreactor, or used to take samples and introduce subsequent portions of dye. I would also like to emphasize that many authors and my earlier observations also indicate that the concentration of biomass has no connection with the removal of dyes.
Comments 9: Did you measure concentration of glucose during your experiments? How does the concentration of dyes influence the carbon source metabolism?
Response 9: Thank you for your question. Glucose levels were not tested. The metabolism of other substrates was not studied, only dyes were focused on, considering this to be the primary goal of the research, especially with such a complex mixture. At the same time, an attempt was made to analyze the spectra using NMR, but the organic content in the samples unfortunately made it impossible to closely observe the changes occurring in the reactors, and therefore this analytical technique was rejected.
Comments 10: Why only 10 g/L? Did you do these experiments with higher concentrations? Can initial glucose concentration influence the adsorption or removal of your dyes?
Response 10: My previous research has shown that the composition of the substrate used is the most optimal for decolorization. Fungi do not need such high concentrations of sugars for their metabolism. At higher concentrations of sugars, the decolorization process was often observed to be inhibited, or at least not to be more effective. Unfortunately, the results of my research on the effect of sugar concentration and other substrate components on the efficiency of decolorization are presented in my book in Polish.
Comments 11: Line 132. Please show those results
Response 111: I have included additional charts in the supplementary materials figures 2-4
Comments 12: Can you prove your claim regarding desorption of dyes? (Line 151)
Response 12: This is a very good question, but even with single dyes, it is difficult to accurately estimate the process. This is the only explanation for the phenomenon in which there is a sudden increase in color in the reactor. It should also be taken into account that the biomass in the reactor was colored, at least most of it. Although over time biomass was also observed that was not colored, the vast majority of the biomass sorbed the dyes. In the case of mixtures, it is even more difficult and this is a subject for detailed research, but not at the stage of batch bioreactors.
Comments 13: Line 168. What is strong biomass? This claim is based on what? Or what kind of observations
Response 13: There it was written very strong mycelium staining. This conclusion was drawn based on visual observations of the color of the biomass. In this case, practically no mycelium hyphae were observed that were not colored.
Comments 14: Line 213. Your tested dyes or products of their degradation are toxic, so my question is: What is the purpose of your research? Decolorization occurred but it is not safe for the environment do discharge this mixture in nature?
Response 15: Thank you for this comment. There are indeed many questions. Samples after the process should be treated as sewage and such a system of assessing their toxicity has been adopted. Separation into individual substances is not only time-consuming but also cost-intensive. Studies for individual substances will never provide a complete answer as to what will happen in the environment. Even in the case of separating substances into individual ones, their effect may be different. In the case of the BWPH strain, further work should be done on optimizing the process in order to be able to reduce the toxicity of sewage in the future, and not only remove the color. However, this requires further research over many years. At the same time, it should be assumed that this is a preliminary stage of treatment, after which the sewage can be directed to a conventional sewage treatment plant where this toxicity can be eliminated. I emphasized this in the conclusions.
Comments 15: Can you show results with individual dyes so one can compare those results to ones with your mixture
Response 15: Thank you very much. I have added two more citations of my own publications, which can show those interested how the process took place for individual dyes. These are items 16-18. However, I did not decide to expand the publication, because one of the reviewers already indicated that the descriptions are too extensive.
Comments 16: Does the mass ratio of dyes influence the degradation or adsorption?
Response 16: Thank you for this question, I am considering this as a new research topic in the future.
Comments 17: Why didn’t you start with lower concentrations and adapt your biomass to such conditions? And then increase the concentration during time?
Response 17: In the case of fungi, the mechanism of dye removal is different than in the case of bacteria, because the process is mainly driven by nonspecific exoenzymes, which can break down compounds with a very complex structure, including a ring structure. Therefore, adaptation to contaminants, in this case dyes, is not necessary, even at different concentrations. The secretion of enzymes is mainly influenced by conditions related to the concentration of C and N.
Comments 18: Line 219. Names in italic
Response 18: Thank you. Done
Comments 19: Line 254. What mechanisms? Explain please
Response 1: Thank you for this question, I added a short note:
“ It was found that the main mechanisms (sorption/biotransformation) used by both strains are different, which determines the stability of decolourisation.”
Reviewer 3 Report
Comments and Suggestions for Authors- This paper is based on the previously published papers and thus materials and method are too simplified and mostly focused on details of explanation of the data.
- The content is redundant and the author need to modify it to more simplified and condensed one.
- The reason why two fungi are selected and characteristics of the two
are necessary.
- Three dyes are selected and decolorisation degrees for three dyes are similar?
- The oxygen supply methods are different , but there was no difference in the decolorization. Dissolved oxygen concentration should be measured to confirm the result.
6.The literature survey especially in recent publications are poor.
Author Response
Comments 1: This paper is based on the previously published papers and thus materials and method are too simplified and mostly focused on details of explanation of the data.
Response 1: Similar studies of a binary mixture of dyes have actually been presented earlier. However, this is the first publication of this type concerning a ternary mixture. It should be emphasized that this is also a publication comparing two strains classified as the same species in different bioreactor operating conditions.
Comments 2: The content is redundant and the author need to modify it to more simplified and condensed one.
Response 2: Thank you for this comment. Other reviewers asked me to expand on certain content of the work instead of condensing it, so I did not take this comment into account, for which I apologize.
Comments 3: The reason why two fungi are selected and characteristics of the two are necessary.
Response 3: Some additional information has been added: The focus was on two different strains of one species to compare not only the efficiency of the process, the effect in the form of toxic or non-toxic impact on the environment because it is difficult to find such a comparison. Although there are many studies on different species of fungi in the process of removing dyes, it is mainly said that a given species removes and another does not remove contaminants. However, little is known about the potential to remove different xenobiotics of individual strains within one species. Therefore, based on previous experiences that showed that the efficiency of removing contaminants may be different for strains from the same species, and having strains isolated from different places, it was decided to make this type of comparison for Pleurotus ostreatus. The additional information:
The BWPH strain was isolated with tissue method from fruit bodies taken from forest near Gliwice, and the K4 strain was isolated by the spore method (fruits collected in Ruda Śląska) [16].
Comments 4: Three dyes are selected and decolorisation degrees for three dyes are similar?
Response 4: Thank you for this question. The dyes were definitely not removed equally. However, looking at the wavelengths at which the measurements were analyzed, the trends were very similar to what was written in lines 129-131 of the original version of the manuscript. Due to the fact that it is difficult to assess what values are assigned to individual dyes at individual wavelengths, only one of the three graphs is presented. Unfortunately, the analysis in the UV range also did not allow determining the changes that occur in the structure of individual dyes. The large amount of organic matter in the sample prevents an in-depth analysis of the problem, which was primarily to determine the effectiveness of the process in various conditions, and to assess the potential impact of the process on the environment.
Comments 5: The oxygen supply methods are different , but there was no difference in the decolorization. Dissolved oxygen concentration should be measured to confirm the result.
Response 5: I agree with this comment, but the tests were conducted in the most sterile conditions possible to check whether this type of reactor can operate with high efficiency. Therefore, it was not decided to introduce a probe that could be a gateway to reactor contamination. Such tests are planned at the stage of increasing the scale of the process and using non-sterile conditions.
Comments 6: The literature survey especially in recent publications are poor.
Response 6: I added some new items: 16, 17, 18, 21, 26
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAuthors have addressed all reviewers remarks and corrected the manuscript accordingly
Reviewer 3 Report
Comments and Suggestions for AuthorsThe paper was properly modified.
