Kinetic Models of Disinfection with Sodium Hypochlorite and Peracetic Acid of Bacteria Isolated from the Effluent of a WWTP

Round 1

Reviewer 1 Report

General

What is the novelty of this study?

There is no compression between the two disinfectants in the discussion and in the conclusion. Which of the disinfectants are better?

The statistical analysis is poor

Line 3 – Title – The abbreviation WWTP should be defined

Abstract

Line 15 – What do you mean by “resistance structures”? The sentence is not clear.

Lines 25 – 27 – Long and unclear sentence.

Introduction

Line 36 – By “protection of living beings” do you mean: protection of human and animals health?

 

Lines 49 – 58 – Dose depends on the disinfectant concentration and the contact time. The values in this paragraph are with concentration units. Please, correct the values, that they will have dose units.

Line 61 – space is missing

Line 62 – which industry?

Line 70 and in all the manuscript – Please, consider changing Ct to CXt or C∙t. In order to differentiate between dose (C∙t) and concentration at time t (Ct).  

Line 86 – space is missing

Lines 89 – 90 – bacteria adapts and develop resistance to disinfection, not disinfection devolve resistance to bacteria

Materials and methods

Lines 118 – 122 – pH, ORP, DO, EC, DTS and TSS where all measured using one electrode or sensor? Please, provide the name of the sensor. Furthermore, it is unconventional to measure TSS using electrode. Maybe, TSS can be measured with light absorbance instrument. Can you provide validation of the TSS measurements?

Lines 184 – 189 – why not use models based on dose rather than concentration? Such as Chick-Watson?

Line 194 – space is missing between the words “the” and “Pearson”

Line 195 – Pearson correlation coefficient is r not R². R² is R square which is coefficient of determination. Furthermore, there are more ways to check fitness of models, and to check linear regression. Please, consider using those methods.

Line 200 – What is statical analysis? Did you mean statistical analysis?

Results and discussion

Table 1 – Please, implement uniformity of reported significant numbers (usually 3 significant numbers are enough e.g. 1.23, 12.3, 123…..). What is PSU? COD and BOD where not mentioned in the methods section.

Line 273 – How Ct - dose required for 4 log removal, were calculated?

Line 274 – again there is a mix between concentration, that has units of mg/L and dose, that has units of mg∙min/L.

Lines 269 – 282 – how come there are more than one Ct (dose required for 4 log removal) reported for each inactivation of bacteria?

Figure 4 and 5 – When did the disinfectant was added? If in t=0, why at that point the concentration is 0? The axis of the graph should start at 0. Negative values do not make sense; Please, correct the caption of the figures; There is no discussion on figure 4 and 5.  

Table 6 – D is not dose it is the initial disinfectant concentration; Please, mention in the footnote of the table that Ct is the dose required to achieve 4 log removal; Are the reported values average? How many repetitions were done for each incubation? Please, add this information to table.

Lines 319 - 331 – flowing this discussion, what was the pH in this study incubations?

Lines 301 – 304 – the literature review presented in those paragraphs, these not refer to the results of this study. Maybe, the paragraphs should be in the introduction.

Line 348 – what “both disinfectants” refer to?

Line 349 – inactivation with chlorine of K. peneumoniae does not have concave shape.

Line 354 – what is DBPs?

Figure 6 and 7 – The axis of the graph should start at 0; Please, correct the captions; What the line represents? Fit? Smoother?

Line 366 – R square is not Pearson coefficient. Furthermore, use statistical tools (goodness of fit etc.) to compare between models, and by plotting the different models and the measured values for compression.

Figure 8 – is missing

Figures 9 – 12 – again, x axis of the graph should start at 0. Model should be plotted using a line (without points). Measured values should be plotted using points (without a line).

The English and grammar of the manuscript is fine. Little rephrasing is needed and misprint need to be corrected

Author Response

General

• What is the novelty of this study?

Currently, wastewater treatment plants WWTPs are facing new challenges, including resistant bacteria to chlorine, the most common disinfection treatment. This work demonstrates this resistance of both Gram-negative and Gram-positive bacteria, which stand out for being endospore formers. In addition, their inactivation by two disinfectants, sodium hypochlorite (NaClO), commonly used around the world, and peracetic acid (PAA), an emerging for wastewater treatment, which represents an alternative to improve the quality of treated water, is evaluated. The bibliography presents data on disinfection kinetics with certified strains; this work is novel because it was possible to identify several species isolated from a real scenario and to use the disinfectants already mentioned.

• There is no compression between the two disinfectants in the discussion and in the conclusion. Which of the disinfectants are better?

The peracetic acid was more effective against Staphylococcus pasteuri and Bacillus subtilis endospores, at the lowest tested concentration (5 mg/L) and contact times of t=10 and 60 minutes respectively, a 99.99% removal (4-log reduction) was achieved. However, NaClO (15 mg/L) was found to be more effective for the inactivation of Klebsiella pneumoniae within 5 minutes. This information has already been added to the manuscript, as well as in the conclusion section.

• The statistical analysis is poor

In this study, the statistical analysis was conducted exclusively to compare the doses of each disinfectant to provide an alternative concentration to use for the elimination of the isolated bacteria. In the case of comparing the concentration of 15 mg/L of both disinfectants, it was not possible to perform the analysis because the inactivation with NaClO was too fast, and the results obtained for Staphylococcus pasteuri and Klebsiella pneumoniae had n<3. Therefore, the data collected from the inactivation itself did not provide sufficient information to conduct a statistical analysis.

• Line 3 – Title – The abbreviation WWTP should be defined

Done

Abstract

• Line 15 – What do you mean by “resistance structures”? The sentence is not clear.

Resistance structures are those that some microorganism present as response to environmental stress, for example, endospores, cysts or oocysts to bacteria or protist respectively. These examples were added.

• Lines 25 – 27 – Long and unclear sentence.

The paragraph was rewritten.

Introduction

• Line 36 – By “protection of living beings” do you mean: protection of human and animals health?

This term has been corrected.

• Lines 49 – 58 – Dose depends on the disinfectant concentration and the contact time. The values in this paragraph are with concentration units. Please, correct the values, that they will have dose units.

The change in concentration per dose was performed.

• Line 61 – space is missing

Done.

• Line 62 – which industry?

Some examples of industries were added.

• Line 70 and in all the manuscript – Please, consider changing Ct to CXt or C∙t. In order to differentiate between dose (C∙t) and concentration at time t (Ct).  

Done

• Line 86 – space is missing

Done

• Lines 89 – 90 – bacteria adapts and develop resistance to disinfection, not disinfection devolve resistance to bacteria

In fact, we were not suggesting the opposite of what the revisor says. Of course, species adapt to the environment and selective pressures such as disinfectants. However, the sentence was modified.

Materials and methods

• Lines 118 – 122 – pH, ORP, DO, EC, DTS and TSS where all measured using one electrode or sensor? Please, provide the name of the sensor. Furthermore, it is unconventional to measure TSS using electrode. Maybe, TSS can be measured with light absorbance instrument. Can you provide validation of the TSS measurements?

pH, ORP, DO, EC, and TDS were measured using an intelligent multiparameter probe from HANNA HI 9828. The DO was measured using a galvanic sensor. As for TSS, it was reported by the WWTP according to the Mexican standard NMX-AA-034-SCFI-2015, which establish the measurement of solids and dissolved salts in natural, wastewater, and treated wastewater, using the gravimetric method.

• Lines 184 – 189 – why not use models based on dose rather than concentration? Such as Chick-Watson?

The Chick models and its modification, the Chick-Watson model, were designed to describe chlorine disinfection. However, they are better suited for inactivation curves with linear trends, which were not observed in the disinfection assays in this study. Additionally, the GinaFit software specializes in fitting curves with shoulder effects, tail effects, or both.

• Line 194 – space is missing between the words “the” and “Pearson”

Done

• Line 195 – Pearson correlation coefficient is r not R2. R2 is R square which is coefficient of determination. Furthermore, there are more ways to check fitness of models, and to check linear regression. Please, consider using those methods.

The coefficient of determination was corrected.

The user manual of GInaFit considers goodness-of-fit indicators such as the mean sum of squared errors, precision indicators like the standard deviation of the data, and variation indicators such as the coefficient of determination to ensure the fitting of the model to the provided experimental data. However, we have chosen to report only the coefficient of determination.

• Line 200 – What is statical analysis? Did you mean statistical analysis?

Done

Results and discussion

• Table 1 – Please, implement uniformity of reported significant numbers (usually 3 significant numbers are enough e.g. 1.23, 12.3, 123…..). What is PSU? COD and BOD where not mentioned in the methods section.

The values of table 1 was corrected. The meaning of PSU has been added.

The values of COD and BOD were obtained by the WWTP according to the NMX-AA-030/1-SCFI-2012 standard, which establishes the measurement of chemical oxygen demand in natural, wastewater, and treated wastewater using the open reflux method, and the NMX-AA-028-SCFI-2005 standard, which establishes the determination of biochemical oxygen demand (BOD₅) in natural, wastewater, and treated wastewater using the iodometric method. The information was added.

• Line 273 – How Ct - dose required for 4 log removal, were calculated?

It was calculated by the product of residual chlorine or peracetic acid and contact time. This information was added.

• Line 274 – again there is a mix between concentration, that has units of mg/L and dose, that has units of mg∙min/L.

Done

• Lines 269 – 282 – how come there are more than one Ct (dose required for 4 log removal) reported for each inactivation of bacteria?

In this work, two concentrations were tested for each disinfectant, the resulting residual varies according to the consumption of the disinfectant so that the C·t value also fluctuates.

• Figure 4 and 5 – When did the disinfectant was added? If in t=0, why at that point the concentration is 0? The axis of the graph should start at 0. Negative values do not make sense; Please, correct the caption of the figures; There is no discussion on figure 4 and 5.  

The disinfectant was added in t= 0. The figures were modified. The discussion of the figures was included.  

• Table 6 – D is not dose it is the initial disinfectant concentration; Please, mention in the footnote of the table that Ct is the dose required to achieve 4 log removal; Are the reported values average? How many repetitions were done for each incubation? Please, add this information to table.

The definition of D was added.

The C·t values in the table do not correspond to the 4-log removal values; they represent the total units of inactivation obtained.

For each species, a disinfection assay was conducted, taking aliquots at different time points. After neutralizing the disinfectant, triplicate decimal dilutions (6 Petri dishes) were plated for microbiological counting. The colony-forming units (CFU) were multiplied by the inverse of the dilution (dilution factor) and the volume plated to obtain the total colonies per plate. The average CFU/mL count meeting the decimal reduction was determined to avoid high standard deviation. The units of inactivation were calculated based on these data.

• Lines 319 - 331 – flowing this discussion, what was the pH in this study incubations?

The pH tested was 7.5 as specified on line 169.

• Lines 301 – 304 – the literature review presented in those paragraphs, these not refer to the results of this study. Maybe, the paragraphs should be in the introduction.

The main emphasis of the study is on highlighting the differences among the three studied bacteria. We believe that it may not be appropriate to include this information in the introduction section.

• Line 348 – what “both disinfectants” refer to?

This phrase was eliminated.

• Line 349 – inactivation with chlorine of peneumoniae does not have concave shape.

The trends of the inactivation curves for this bacterium best fit the Weibull model which refers to a concave shape.

• Line 354 – what is DBPs?

Abbreviation added on line 58

• Figure 6 and 7 – The axis of the graph should start at 0; Please, correct the captions; What the line represents? Fit? Smoother?

The graphs have been changed. Dots represents experimental data and the inactivation curves are represented by smooth lines.

• Line 366 – R square is not Pearson coefficient. Furthermore, use statistical tools (goodness of fit etc.) to compare between models, and by plotting the different models and the measured values for compression.

The coefficient of determination was corrected. The explanation was already given as equal in the comment on line 195.

• Figure 8 – is missing

It is located on line 402

• Figures 9 – 12 – again, x axis of the graph should start at 0. Model should be plotted using a line (without points). Measured values should be plotted using points (without a line).

The graphs have been changed.

Reviewer 2 Report

The manuscript presented a study on Kinetic models of disinfection with sodium hypochlorite and peracetic acid of bacteria isolated from the effluent of a WWTP. In general, the topic is very interesting and relevant. However, the reviewer found some critical concerns on the results presenting, manuscript writing, and especially, the discussion of the findings.   1) In table 1, BOD is BOD5 or BOD20? The relationship between all parameters in the table and subsequent disinfection needs to be discussed. 2) Add a discussion section, in-depth discussing the findings, the critical viewpoints and logical building up and the meaning of the work can be reflected by the discussion. 3）It is suggested removing Tables 2-4 as they are not closely related to the research topic of the paper. 4）Analyze the reasons for the different disinfection kinetic models when using different disinfectants. 5）The conclusion section does not fully reflect the research content, and it is recommended to rewrite it.  

Please check carefully the grammar and sentences.

Author Response

• In table 1, BOD is BOD5 or BOD20? The relationship between all parameters in the table and subsequent disinfection needs to be discussed. 

We refer a BOD₅, it was corrected. The physicochemical parameters obtained were suitable for the isolation of the microorganism. This information was added.  

• Add a discussion section, in-depth discussing the findings, the critical viewpoints and logical building up and the meaning of the work can be reflected by the discussion. 

We consider that the sections of the article were structured for a better reading comprehension, we do not believe it necessary to separate the discussions. In the corrected manuscript, information was added in lines 290 - 295 and 298-302 to expand the discussion. In addition, for example, the explanation of the calculation of C·t was added, we refer to doses instead concentrations. We made more emphasis on the conclusions.

• It is suggested removing Tables 2-4 as they are not closely related to the research topic of the paper. 

As mentioned, this work is pioneering for presenting results of bacteria isolated from a WWTP, the characterization and identification of these is necessary to support that different species were found. Traditional microbiology techniques continue to be a tool that supports modern, rapid, and innovative techniques such as MALDI Biotyper to confirm which species we are working with.

• Analyze the reasons for the different disinfection kinetic models when using different disinfectants. 

Although the objective of disinfectants is to inactivate microorganisms, each one has a different effect on cells or different resistance structures such as endospores, cysts or oocysts. In addition, the mechanism of action on cells, concentration, contact time and dosage, it seemed relevant to us to consider the comparison between the two most used disinfectants and above all to be pioneers in obtaining kinetic models of disinfection for bacteria isolated from a real scenario, unlike those reported with certified strains in numerous investigations.

• The conclusion section does not fully reflect the research content, and it is recommended to rewrite it.  

This section was rewritten.