The Use of Chitosan for Flocculation Recovery of Bacillus Biomass Grown on Dairy and Wine Industry Effluents

Round 1

Reviewer 1 Report

The Introduction section must describe a hypothesis that should be confirmed or refuted in Conclusion section.

Line 148. Give an explanation to «аlpha»

Table 1 and 2. Set the experiments in the order of increasing or decreasing of the inhibition zone.

Table 7. Give explanations to «ss» and «ms» and other related abbreviations.

Author Response

Reviewer 1

 

Point 1: The introduction section must describe a hypothesis that should be confirmed or refused in Conclusion section.

Response 1: The aim of the study is better defined, and in the conclusion section the confirmation of the hypothesis about flocculation and synergetic effect of chitosan are added.

 

Point 2: Give an explanation to «alpha»

Response 2: The explanation is added to the manuscript as follows.

“In a central composite design, alpha is the distance between each axial point (also known as a star point) and the center. Each variable in the design has new extreme values (low and high) represented by the star points.”

 

Point 3: Table 1 and 2. Set the experiments in order of increasing or decreasing of the inhibition zone.

Response 3: The tables are arranged in way to typical presentation of the central composite design for two factors, i.e. standard order of experiments was applied. Also, we had three responses and authors think that it is the best to keep the current arrangement in order not to follow the trend of just one response.

 

Point 4: Table 7. Give explanations to «ss» and «ms» and other related abbreviations.

Response 4: the subsection “2.4.1. Statistical Analysis” is added in Materials and Methods, where abbreviations are explained.

 

The authors would like to thank the reviewer 1 for the thoughtful comments and suggestions and efforts made to improve our manuscript. We tried our best to address the general and specific concerns the reviewer expressed. We hope we were successful in upgrading the quality of the manuscript according to the abovementioned instructions.

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

Reviewer 2

 

Point 1: This study presents a methodical and rigorous approach to evaluate the effectiveness of chitosan flocculation in recovering bacterial biomass from liquid waste of dairy and wine industry. The results of the study demonstrate that chitosan can be an effective flocculation agent for biomass recovery in these effluents, with optimal values for flocculation, settling velocity, and antimicrobial activity. The use of response surface methodology and multi-objective optimization helped to identify the optimal experimental conditions to maximize the desired outcomes. The validation results of the experimental predictions confirmed the validity of the models used in this study. In summary, this study provides useful information on the use of chitosan as a flocculation agent for bacterial biomass recovery from liquid waste of dairy and wine industry. The results presented in this study can be useful for implementing sustainable practices in these industries by reducing their environmental impact while improving their operational efficiency. However, certain modifications are required, particularly in certain sections.

Response 1: The authors would like to thank the reviewer for these comments. We tried our best to address all general and specific comments.

 

Point 2: Here are some suggestions for improving the Abstract: Start with a clear statement of the research objective and the main findings. Provide some context on why this research is important and how it contributes to existing knowledge in the field. Use more specific and descriptive language to explain the methods and results. For example, instead of simply stating that "Confirmation experiments for both effluents at the optimum experimental values gave satisfactory agreement between predicted and experimental response values," you could explain what these experiments entailed and how they confirmed the validity of the findings. Consider adding a sentence or two on the potential implications or applications of these findings.

Response 2: The abstract is modified in accordance to the suggestions.

The downstream processing of efficient biomass-based microbial biopesticides is heavily reliant on getting the largest concentration of viable cells in the most cost-effective manner. The goal of this research was to assess the ability of chitosan flocculation to recover bacterial Bacillus sp. BioSol021 biomass from the broth after biological treatment of wastewaters from dairy and wine industry. Second order factorial design models were used to estimate the effect of chitosan concentration and mixing speed on flocculation efficiency, settling velocity and antimicrobial activity against Aspergillus flavus i.e. inhibition zone diameter. Response surface methodology was followed by multi-objective optimization by applying desirability function (DF), and genetic algorithm (GA). The optimum values for flocculation efficiency, settling velocity and inhibition zone diameter for cheese whey effluent were 88%, 0.10 mm/s and 51.00 mm, respectively. In the case of winery flotation effluent the optimum values were flocculation efficiency 95%, settling velocity 0.05 mm/s while the inhibition zone diameter was 48.00 mm. These results indicate that utilizing chitosan as a flocculation agent not only fits the criteria for effective downstream processing, but also has a synergistic effect on Bacillus sp. antibacterial activity.

 

Point 3: Materials and methods I highly recommend including a statistical analysis section when comparing the results of various experiments.

Response 3: The comment was acknowledged, and changes were made according to the suggestion. The subsection “2.4.1. Statistical Analysis” is added in Materials and Methods as follows.

2.4.1. Statistical Analysis

Using multiple regression analysis on the experimental data, the quadratic equation was defined to relate responses and design variables. Statistical analysis of the quadratic polynomial model was performed by the analysis of variance (ANOVA) to check models adequacy.

ANOVA consists of calculations that provide information about levels of variability within a regression model and form a basis for tests of significance. ANOVA has a number of parameters, which are summarized in a table, the degrees of freedom (df), sum of squares (SS) and mean square (MS). These parameters are calculated for model and error. Tests of significance are conducted by analyzing p-values.

The p-values obtained are used to assess the statistical significance of the models and their coefficients, while the quality of the experimental data fitting is estimated using the Lack-of-fit, Pure error, and R2 (coefficient of determination) values. The coefficient of determination value can be defined as the proportion of variability around the mean for the dependent variable that the model can account for, in range between 0 and 1 (the ideal fit). Due to measurement errors or relationships between responses and factors that the selected model cannot describe, a selected second-order polynomial model cannot fit the measured values perfectly. Actually, this fact causes deviations between predicted and measured values, which are referred to as residuals. At the design points, therefore, so-called residual values exist [12].

The significance of the lack of fit can also be determined, where some runs are rep-licated, such as central composite design. A statistical test based on splitting the residual error sum of squares into two components: lack-of-fit sum of squares (associated with variation due to factors other than measurement error) and pure error sum of squares (associated with random variation caused by measurement error) is used to evaluate the model's acceptability. A low p-value, below 0.05, for lack-of-fit in the ANOVA table shows that the analyzed model does not adequately fit the experimental data [12, 23]. All statistical analyses were performed at the significance level of 95%.

 

Point 4: Conclusion part: The conclusion provides a comprehensive summary of the study's findings and the optimization process using chitosan as a natural flocculant for biomass recovery. However, the conclusion could be improved by including some practical implications of the research. For example, the study could highlight how the use of chitosan as a flocculation agent can lead to cost savings and environmental benefits for the dairy and wine industry. Additionally, the conclusion could suggest possible future research directions, such as investigating the scalability of the chitosan flocculation process to industrial-scale applications. Finally, it could be useful to include a brief discussion on the limitations of the study and the potential areas for improvement to further optimize the bacterial harvesting method using chitosan.

Response 4: The comment was acknowledged, and changes were made in revised text according to the suggestion.

 

The authors would like to thank the reviewer 2 for the thoughtful comments and suggestions and efforts made to improve our manuscript. We tried our best to address the general and specific concerns the reviewer expressed. We hope we were successful in upgrading the quality of the manuscript according to the abovementioned instructions.

Reviewer 3 Report

This manuscript discusses the ability of chitosan flocculation to recover Bacillus sp. BioSol021 biomass from the broth of both cheese whey and winery flotation wastewaters. The optimization process for both cases are conducted, and the results are discussed clearly. 

I have general comments that may help in improving the manuscript:

- Abstract: add a concluding remark at the end, after the results.

- Introduction needs improvements:

- Line 30-34: "Aflatoxin B1 (AFB1) is....as the most potent natural carcinogen [5, 6]." is not related to the topic.

-  Detailed information on biopesticides are far from the manuscript's objective. Instead, add the characteristics of the wastewaters, some statistics on the production and effluent generated. What is the current treatment method, and why is chitosan flocculation is a better choice?

-Line 221, discuss Figure 1(a) before Figure 1(b).

- The conclusion part is too long, summarize the main findings and their importance in the field, no need to repeating the results and discussing them.

 

 

Author Response

Reviewer 3

 

Point 1: This manuscript discusses the ability of chitosan flocculation to recover Bacillus sp. BioSol021 biomass from the broth of both cheese whey and winery flotation wastewaters. The optimization processes for both cases are conducted, and the results are discussed clearly. I have general comments that may help in improving the manuscript.

Response 1: The authors would like to thank the reviewer for these comments. We tried our best to address all general comments.

 

Point 2: Abstract: add a concluding remark at the end, after the results.

Response 2: The abstract is corrected as suggested.

 

Point 2: Introduction needs improvements: Line 30-34: "Aflatoxin B1 (AFB1) is....as the most potent natural carcinogen [5, 6]." is not related to the topic. Detailed information on biopesticides are far from the manuscript's objective.

Response 2: Within this research, the optimization of the flocculation method was performed aiming the maximization of microbial separation efficiency, as well as the synergistic effects of Bacillus biomass and chitosan in term of antimicrobial activity. We applied “from flocculation to formulation” concept aimed at the increase of the sustainability of the downstream processing, as important part of biocontrol agents production technology. This study confirmed the synergistic effects of the Bacillus sp. BioSol021 and chitosan in term of biocontrol activity against aflatoxigenic Aspergillus flavus isolate. Considering the purpose of the final product, we think it is important to emphasize its application as a biocontrol agent and its potential in suppressing the production of aflatoxin. On the other hand, if the reviewer considers it necessary to shorten the mentioned parts from the Introduction section, we are at our disposal to do so to an appropriate extent.

 

Point 3: Instead, add the characteristics of the wastewaters, some statistics on the production and effluent generated. What is the current treatment method, and why is chitosan flocculation is a better choice?

Response 3: The mentioned topics were covered in detail in our previous publication that dealt with the upstream process. In order not to repeat ourselves, we have cited this work within the Material and methods section:

[4] Dmitrović, S.; Pajčin, I.; Vlajkov, V.; Grahovac, M.; Jokić, A.; Grahovac, J. Dairy and wine industry effluents as alternative media for the production of Bacillus-based biocontrol agents. Bioengineering 2022, 9, 663. https://doi.org/10.3390/bioengineering9110663

 

Point 4: Line 221, discuss Figure 1(a) before Figure 1(b).

Response 4: Corrected in the manuscript.

 

Point 5: The conclusion part is too long; summarize the main findings and their importance in the field, no need to repeating the results and discussing them.

Response 5: The reviewer’s comment was acknowledged, and changes were made according to the suggestion.

 

The authors would like to thank the reviewer 3 for the thoughtful comments and suggestions and efforts made to improve our manuscript. We tried our best to address the general and specific concerns the reviewer expressed. We hope we were successful in upgrading the quality of the manuscript according to the abovementioned instructions.