Microbial Culture Condition Optimization and Fiber Reinforcement on Microbial-Induced Carbonate Precipitation for Soil Stabilization

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The topic looks interesting. However I have the following comments

1- The paper title should be changed. The paper focuses more on microbial culture conditions more than soil stabilization. That is very clear from the discussion of the results

2- The author should state clearly the time of testing after sample preparation since there are changes takes place in the mixture

3- The grain size distribution curve should be presented instead of table 1

4- The author stated that the ductility improved. The is no stress strain curve in the manuscript to conclude that

5- Just two simple test were conducted UC and Permeability test. This will make this work short of full paper  

 

Author Response

Reviewer #1:

1. The paper title should be changed. The paper focuses more on microbial culture conditions more than soil stabilization. That is very clear from the discussion of the results.

Response: Thank you for your suggestion. Based on your advice, in order to highlight the focus of the research, the title of the paper has been changed to " Microbial Culture Condition Optimization and Fiber Reinforcement on Microbial-Induced Carbonate Precipitation for Soil Stabilization."

The above information has been added to the revised manuscript.

2. The author should state clearly the time of testing after sample preparation since there are changes takes place in the mixture.

Response: Thank you for your feedback. The process from sample preparation to performance testing can be summarized in the following three steps:

• Circulatory infusion of bacterial solution and cementation solution into the sand sample within the mold.
• After infusion, place the sample in a drying oven set at 60°C for 24 hours to dry.
• After drying, perform the performance testing.

The performance testing is conducted after the sample has been dried for 24 hours. The test time is now added to section 2.3, which is as follows:

“ The samples, after completing the grouting process, are placed in an oven and dried at a temperature of 60°C for 24 hours. Once dried, the samples are ready for performance testing.”

The above information has been added to the revised manuscript.

3. The grain size distribution curve should be presented instead of table 1.

Response: Thank you for your suggestion. The grain size distribution curve of the sand is shown in Figure 1.

Figure 1. Particle size distribution curve.

The above information has been added to the revised manuscript.

4. The author stated that the ductility improved. The is no stress strain curve in the manuscript to conclude that.

Response: Thanks for pointing out this problem. The improvement of fiber incorporation in addressing the fracture brittleness of microbially cemented sandy soil can be reflected in the failure morphology of the cemented sandy soil column samples under applied stress. By observing the failure morphology of these samples, it is evident that the inclusion of fibers significantly enhances the ductility and integrity of the microbially cemented sandy soil.

The failure morphology of cemented sandy soil column samples under different fiber content conditions has been added to Section 3.3. The specific content is as follows:

“Figure 10 shows the failure morphology of the samples from the group with no fiber content during the unconfined compressive strength test. It is evident from the figure that the failure mode is typical of brittle failure. During the failure process, due to the applied external force, cracks first appear on the surface of the sandy soil column, forming fracture surfaces and causing localized failure. As the external force continues to increase, the collapse caused by the localized failure extends downward until the entire column becomes unstable and the original structure is completely destroyed. Throughout the entire loading process, sand particles continue to detach due to the formation of fracture surfaces. After brittle failure, the sandy soil column exhibits an irregular failure surface, with large depressions and protrusions. The residual sandy soil after failure becomes loose, with the once relatively compact sand particles dispersing due to the fracture. The remaining sandy soil is seen as scattered individual sand particles, fragments, or clumps.

Figure 11 depicts the failure morphology of the samples from the experimental group with 0.6% fiber content during the unconfined compressive strength test. Unlike the sudden fracture of brittle failure, the failure mode here involves more noticeable deformation, characteristic of ductile failure. During the failure process, small cracks first appear in localized areas of the sandy soil column. As the stress increases, these cracks expand along multiple paths, gradually forming larger fissures. The sandy soil gradually loosens, but no rapid collapse occurs. Under the tensile force of the fibers, the sandy soil undergoes a slower failure process. Throughout the failure, the specimen experiences compressive deformation due to the external force. Due to the presence of fibers, the surface of the specimen after failure exhibits a relatively uniform crack distribution, without distinct fracture surfaces. Additionally, the residual sandy soil structure after failure retains a certain degree of integrity, not completely disintegrating, and the sand particles do not experience significant detachment. The main reason fiber incorporation changes the failure mode of the soil from brittle failure to ductile failure is that fibers, by connecting and reinforcing the bonds between soil particles, effectively suppress the rapid propagation of cracks and slow down the crack growth rate. This results in a slower and more gradual development of cracks, preventing the sudden collapse characteristic of brittle failure. On the other hand, the fiber-reinforced soil can disperse stress in multiple directions, forming the parallel development of multiple micro-cracks, which further reduces the brittle fracture caused by the rapid expansion of a single crack.”

Figure 10. Failure morphology of solidified sandy soil column samples without fiber content. (a) Failure process. (b) Residual morphology

 

Figure 11. Failure morphology of solidified sandy soil column sample with fiber content of 0.6%. (a) Failure process. (b) Residual morphology

The above information has been added to the revised manuscript.

5. Just two simple test were conducted UC and Permeability test. This will make this work short of full paper

Response: Thank you for your insightful comments. We understand your concern regarding the number of tests conducted in our study. We would like to emphasize that our research aims to reveal the synergistic mechanism between MICP and fiber reinforcement in soil stabilization, which requires a focused but comprehensive approach. The UCS and permeability tests were carefully chosen as they directly reflect the strength development and durability of the MICP-treated soil, which are critical parameters for practical engineering applications.

To complement these macroscopic tests, we conducted XRD and SEM-EDS analyses to elucidate the microstructural evolution and mineralization efficiency of MICP in fiber-reinforced soils. These microstructural characterizations provide deeper insights into the formation and distribution of calcium carbonate precipitation, further validating our conclusions.

We have acknowledged the need for further investigations, such as long-term durability studies and field-scale validations, in our future research plans. We sincerely appreciate your valuable feedback, which has allowed us to strengthen our future studies.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Comments for Author:

I have thoroughly reviewed this paper, titled “Enhanced Soil Stabilization via Microbial-Induced Carbonate Precipitation with Polyethylene Fiber Reinforcement.” The author argues that conventional soil stabilization techniques, including cement and chemical grouting, are not only energy-intensive but also detrimental to the environment. This article offers valuable insights and addresses highly relevant topics in the field of sustainable agriculture. Thus, I recommend it for publication in this journal. However, I suggest that certain revisions and corrections be made prior to its final acceptance.

1. I believe the title is appropriate, but if possible, I recommend revising it to ensure it is more easily understood by readers.
2. Line 13-14: MICP-treated soils often exhibit low mineralization efficiency and brittleness. Revise the sentence.
3. The abstract currently lacks details regarding the methods and materials section. It would be beneficial to include basic information about the experiment, such as the experimental design, and duration, to provide readers with a clearer understanding of the study.
4. Remove the abbreviation from the Keywords.
5. Lines 33-37: Please provide references to support this statement
6. Line 75: The term “Microbial-Induced Carbonate Precipitation (MICP)” is already abbreviated in the abstract, so it should not be abbreviated again in the introduction section. Instead, it can be introduced in its full form at the first mention in the introduction, followed by the abbreviation for subsequent references.
7. Lines: 75-77: Please provide references to support this statement.
8. Lines 175-275: Provides a reference for each method used in this experiment.
9.  Lines 278-279: In MICP technology, urea plays a crucial role by providing essential nutrients for microbial growth and promoting the generation of carbonate ions [40, 41]”. Please do not describe this statement in the results section. Just pasted your findings in this section. Remove the sentence.
10. Lines 293-294: This extreme alkalinity suppresses mi- 292 microbial metabolic activity and growth and may also interfere with protein synthesis and enzyme activity, thereby impacting the microorganism’s normal physiological functions [44-46]. Just pasted your findings in this section. Remove the sentence, or subjeced in the introduction or in the discussion section.
11. I noticed that several references are included in the results section. Please remove them from this section and discuss them in their appropriate context, such as the introduction or discussion sections.
12. Please include references to support the explanations and contextualize your findings in the discussion section. This will strengthen the validity and credibility of your arguments.
13. Please carefully revise the entire manuscript by eliminating double spaces, enhancing the English language for clarity, and ensuring the manuscript is according to the journal format.

     

     

 

 

 

 

Comments on the Quality of English Language

Please carefully revise the entire manuscript by eliminating double spaces, enhancing the English language for clarity, and ensuring the manuscript is according to the journal format.

 

Author Response

1. I believe the title is appropriate, but if possible, I recommend revising it to ensure it is more easily understood by readers.

Response: Thank you for your suggestion. Based on your advice, to ensure better understanding for the readers, the title of the paper has been changed to " Microbial Culture Condition Optimization and Fiber Reinforcement on MICP for Soil Stabilization."

The above information has been added to the revised manuscript.

2. Line 13-14: MICP-treated soils often exhibit low mineralization efficiency and brittleness. Revise the sentence.

Response: Thank you for your feedback. To ensure better understanding for the readers, I have made adjustments to this sentence. The specific content is as follows:

“However, the application of MICP technology in soil stabilization still faces certain challenges. First, the mineralization efficiency of microorganisms needs to be improved to optimize the uniformity and stability of carbonate precipitation, thereby enhancing the effectiveness of soil stabilization. Second, MICP-treated soil generally exhibits high fracture brittleness, which may limit its practical engineering applications. Therefore, improving microbial mineralization efficiency and enhancing the ductility and overall integrity of stabilized soil remain key issues that need to be addressed for the broader application of MICP technology.”

The above information has been added to the revised manuscript.

3. The abstract currently lacks details regarding the methods and materials section. It would be beneficial to include basic information about the experiment, such as the experimental design, and duration, to provide readers with a clearer understanding of the study.

Response: Thank you for pointing out the issue. I have now included the detailed information about the methods and materials section in the abstract. The specific content is as follows:

“This study addresses these challenges by optimizing microbial culture conditions and incorporating polyethylene fiber reinforcement. The experiments utilized sandy soil and polyethylene fibers, with Bacillus pasteurii as the microbial strain. The overall experimental process included microbial cultivation, specimen solidification, and performance testing. Optimization experiments for microbial culture conditions indicated that the optimal urea concentration was 0.5 mol/L and the optimal pH was 9, significantly enhancing microbial growth and urease activity, thereby improving calcium carbonate production efficiency. Specimens with different fiber contents (0% to 1%) were prepared using a stepwise intermittent grouting technique to form cylindrical samples. Performance test results indicated that at a fiber content of 0.6%, the unconfined compressive strength (UCS) increased by 80%, while at a fiber content of 0.4%, the permeability coefficient reached its minimum value (5.83 × 10⁻⁵ cm/s). Furthermore, microscopic analyses, including X-ray diffraction (XRD) and scanning electron microscopy with energy-dispersive spectroscopy (SEM-EDS), revealed the synergistic effect between calcite precipitation and fiber reinforcement.“

The above information has been added to the revised manuscript.

4. Remove the abbreviation from the Keywords.

Response: Thank you for your feedback. I have now removed the abbreviations from the keywords

5. Lines 33-37: Please provide references to support this statement

Response: Thank you for your suggestion. I have added the relevant references to support this statement. The specific content is as follows:

“Effective soil stabilization helps mitigate the adverse effects of soil erosion, improves soil fertility, and supports the rehabilitation of degraded lands, thereby enhancing ecosystem health and resilience ^[1]. By stabilizing soils, we can prevent further degrada-tion of valuable land resources, safeguard biodiversity, and promote the sustainable management of natural habitats, which are essential for the well-being of both human beings and our planet ^[2].”

The above information has been added to the revised manuscript.

6. Line 75: The term “Microbial-Induced Carbonate Precipitation (MICP)” is already abbreviated in the abstract, so it should not be abbreviated again in the introduction section. Instead, it can be introduced in its full form at the first mention in the introduction, followed by the abbreviation for subsequent references.

Response: Thank you for your feedback. I have made revisions for this issue and checked the consistency of abbreviation usage throughout the entire text.

7. Lines: 75-77: Please provide references to support this statement.

Response: Thank you for your suggestion. I have added the relevant references to support this statement. The specific content is as follows:

“Microbial-Induced Carbonate Precipitation (MICP) emerges as an innovative, eco-friendly alternative that uses biological processes to enhance soil properties while aligning with global commitments to sustainable development and environmental restoration ^{[3] [4]}.”

The above information has been added to the revised manuscript.

8. Lines 175-275: Provides a reference for each method used in this experiment.

Response: Thank you for your suggestion. The references for the relevant testing methods used in this experiment have now been added to the article, with the details as follows:

• Microbial activity test

The urease activity in the experiment was measured using the conductivity method proposed by Whiffin^[5].

• Calcium carbonate production rate

The experiment employed the acid washing method^[6] to determine the calcium carbonate production.

• Unconfined compressive strength

The unconfined compressive strength test was conducted following the Standard for Geotechnical Testing Method (GB/T 50123-2019)^[7].

• Permeability

The permeability test was conducted based on the principles of the variable head permeability test outlined in the Standard for Geotechnical Testing Method (GB/T 50123-2019) ^[7] . A simplified permeability measurement device was designed to evaluate the permeability of the specimens.

The above information has been added to the revised manuscript.

9. Lines 278-279: In MICP technology, urea plays a crucial role by providing essential nutrients for microbial growth and promoting the generation of carbonate ions [40, 41]”. Please do not describe this statement in the results section. Just pasted your findings in this section. Remove the sentence.

Response: Thank you for your suggestion. I have removed this statement from the results section.

10. Lines 293-294: This extreme alkalinity suppresses mi- 292 microbial metabolic activity and growth and may also interfere with protein synthesis and enzyme activity, thereby impacting the microorganism’s normal physiological functions [44-46]. Just pasted your findings in this section. Remove the sentence, or subjeced in the introduction or in the discussion section.

Response: Thank you for your suggestion. I have removed this statement from the results section.

11. I noticed that several references are included in the results section. Please remove them from this section and discuss them in their appropriate context, such as the introduction or discussion sections.

Response: Thank you for your feedback. I have adjusted the structure of the results and discussion sections and made corresponding revisions to the content. The revised content can be found in lines 302-435 of the revised manuscript..

12. Please include references to support the explanations and contextualize your findings in the discussion section. This will strengthen the validity and credibility of your arguments.

Response: Thank you for your suggestion. To strengthen the effectiveness and credibility of the arguments in the paper, I have now added supporting references to the discussion section. The revised content can be found in lines 435-596 of the revised manuscript.

13. Please carefully revise the entire manuscript by eliminating double spaces, enhancing the English language for clarity, and ensuring the manuscript is according to the journal format.

Response: Thank you for your suggestion. Based on your advice, I have carefully revised the entire manuscript to improve the clarity of the English language and ensured that the manuscript complies with the journal's format.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The study presents an innovative and environmentally friendly approach to soil stabilization, combining MICP technology with fiber reinforcement to enhance mechanical performance. The comprehensive experimental design, including the optimization of microbial growth conditions and the integration of fiber reinforcement, provides valuable insights for future applications in geotechnical engineering. The results clearly demonstrate the effectiveness of this method in improving soil strength and reducing permeability, which could have significant implications for stabilization of infrastructure foundation. However, I have a few recommendations to further strengthen the manuscript:

General comments:

1. The experimental design is generally well-structured, but there is a lack of detail regarding the reproducibility of the experiments and error control. The number of samples tested under each condition and the number of repetitions should be explicitly stated. Additionally, a description of how experimental errors were controlled would improve the methodological rigor.

2. The study lacks a screening process for Bacillus pasteurii. A preliminary identification of the bacterial strain used would enhance the scientific rigor and reliability of the experimental results. Consider adding a bacterial strain characterization section in the materials and methods.

3. Ensure consistency in the use of technical terminology throughout the manuscript. Additionally, check that all figure labels, axis units, and variables are clearly defined and appropriately formatted.

Specific comments:

1. In the Section of Materials and Methods, the choice of polyethylene fiber is not sufficiently justified. It would be beneficial to explain why polyethylene was selected over other fiber types and how its properties contribute to improved soil performance.

2. In Table 3, the fiber content is presented in percentage terms, but it is unclear whether this refers to mass or volume fraction. Please clarify in the materials and methods section.

3. The results on UCS and permeability coefficient are clearly presented, but the discussion on why strength decreases when fiber content exceeds 0.6% is insufficient. How does fiber agglomeration contribute to increased porosity and reduced strength? A more in-depth explanation would strengthen the discussion.

4. In the Section 3.1, the microbial growth curve is an essential parameter for optimizing MICP treatment. It is recommended to include a growth curve for Bacillus pasteurii under optimal conditions to provide a clearer understanding of bacterial activity over time.

Author Response

Reviewer #3:

General comments:

1. The experimental design is generally well-structured, but there is a lack of detail regarding the reproducibility of the experiments and error control. The number of samples tested under each condition and the number of repetitions should be explicitly stated. Additionally, a description of how experimental errors were controlled would improve the methodological rigor.

Response: Thank you for your suggestion. The number of samples tested under different conditions and the number of repetitions are as follows:

• UCS

A total of 18 specimens were used for the UCS tests, with 6 variable groups of fiber content ranging from 0% to 1%, each consisting of 3 specimens. The UCS results for each experimental group are represented by the average of the three test results.

• Permeability

A total of 18 specimens were used for permeability testing, with 6 variable groups of fiber content ranging from 0% to 1%, each consisting of 3 specimens. The permeability coefficient results for each experimental group are represented by the average of the three test results.

The above information has been added to the revised manuscript.

2. The study lacks a screening process for Bacillus pasteurii. A preliminary identification of the bacterial strain used would enhance the scientific rigor and reliability of the experimental results. Consider adding a bacterial strain characterization section in the materials and methods.

Response: Thank you for your insightful comments. To improve the scientific rigor and reliability of the experimental results, the strain screening process has been added. Preliminary identification of Bacillus pasteurii was performed using an inverted biological microscope (IX83, OLYMPUS) and Gram staining technique. The observation and identification results (Figure 2) are consistent with the characteristics of Bacillus pasteurii, thereby confirming that the microbial activity occurring in subsequent experiments is predominantly driven by Bacillus pasteurii.

The identification and selection of Bacillus pasteurii used in the experiment have now been added to Section 2.1.2. The specific content is as follows:

“ The Bacillus pasteurii cultured in the experiment was initially identified using an in-verted biological microscope (IX83, OLYMPUS) and Gram staining technique. The ob-servation results (Figure 2) were consistent with the characteristics of Bacillus pasteur-ii.”

Figure 2. Microscopic observation of Bacillus bussiae in Gram staining state.

The above information has been added to the revised manuscript.

3. Ensure consistency in the use of technical terminology throughout the manuscript. Additionally, check that all figure labels, axis units, and variables are clearly defined and appropriately formatted.

Response: Thank you for your feedback. To ensure the rigor of the paper, I have reviewed and made appropriate revisions to ensure consistency in the use of technical terms, accuracy in figure labels, axis units and variable definitions, as well as uniformity in formatting throughout the manuscript.

Specific comments:

1. In the Section of Materials and Methods, the choice of polyethylene fiber is not sufficiently justified. It would be beneficial to explain why polyethylene was selected over other fiber types and how its properties contribute to improved soil performance.

Response: Thank you very much for your suggestion. The fiber used in the experiment is polyethylene fiber. Polyethylene fiber was chosen as the reinforcement material for this study primarily due to its excellent chemical stability, high tensile strength, good resistance to acids and alkalis, and relatively low cost. Compared to other fiber types (such as polypropylene fiber, glass fiber, and cotton-linen fiber), polyethylene fiber demonstrates better durability and anti-aging performance in soil environments, making it well-suited for integration with MICP technology. Its high tensile strength and elastic modulus help to evenly distribute stress and reduce localized stress concentrations, thereby improving the compressive strength and crack resistance of the soil.

The explanation regarding the reasons for selecting polyethylene fiber and how its properties help improve soil performance has now been added to Section 2.1.1. The content is as follows:

"The polyethylene fiber chosen for the experiment is a synthetic fiber, which, compared to natural fibers and other fibers (such as polypropylene fiber and glass fiber), exhibits excellent chemical stability and high tensile strength. The good chemical stability of polyethylene fiber provides better durability and anti-aging performance in soil environments, ensuring its long-term stability within the soil matrix, making it suitable for integration with MICP technology. Additionally, the high tensile strength and elastic modulus of polyethylene fiber help to improve the soil's ductility, evenly distribute stress, and reduce localized stress concentrations, thereby enhancing the compressive strength and crack resistance of the soil, addressing the brittleness issue in MICP-treated soils."

The above information has been added to the revised manuscript.

2. In Table 3, the fiber content is presented in percentage terms, but it is unclear whether this refers to mass or volume fraction. Please clarify in the materials and methods section.

Response: Thanks for pointing out this problem. The meaning of the percentage for fiber content in Table 3, which refers to the percentage of the sand soil volume. The meaning of the percentage in this context has now been added to Section 2.2. The content is as follows:

“The meaning of the percentage for fiber content in Table 3, which refers to the per-centage of the sand soil volume.”

The above information has been added to the revised manuscript.

3. The results on UCS and permeability coefficient are clearly presented, but the discussion on why strength decreases when fiber content exceeds 0.6% is insufficient. How does fiber agglomeration contribute to increased porosity and reduced strength? A more in-depth explanation would strengthen the discussion.

Response: Thank you for your insightful comments. The increase in porosity caused by fiber agglomeration is due to the inability of the fibers to distribute uniformly and effectively within the soil. The interlacing and overlapping of fibers form larger and more irregular void structures, while also reducing the contact and filling between the soil particles and fibers. This significantly undermines the role of fibers in enhancing the strength of the soil and may even render the soil’s mechanical properties more fragile. A more detailed explanation of the mechanism by which fiber agglomeration increases porosity and decreases strength has been added to Section 4.3. The specific content is as follows:

“When fibers agglomerate in the soil, they interlace and overlap with each other, preventing the fibers from dispersing effectively within the soil and leading to the formation of irregular void structures. These voids are not the pores formed by soil particles but are structural voids created by fiber agglomeration. The structure formed after fiber cohesion is typically irregular and may contain some larger voids. These voids are not individual pores but a porous structure resulting from fiber agglomeration. This structure may include larger gaps and irregular channels, which increase the overall porosity. The size and shape of these irregular voids can vary significantly, leading to a non-uniform increase in soil porosity. Additionally, under normal conditions, fibers may fill the gaps between soil particles, increasing the compaction of the soil. However, when fibers agglomerate, the contact area between the fibers and soil particles is reduced, causing the fibers to be less effective at connecting with the soil particles compared to when they are individually distributed. As a result, the fibers are unable to efficiently fill the soil’s pores, leading to an increase in porosity. The larger voids and pores in fiber agglomeration areas make these regions weak points within the soil ^{[8, 9]}. When subjected to external forces, these agglomerated regions create stress mismatch zones with the surrounding soil. Such areas are more prone to localized failure or crack propagation, which can lead to a reduction in overall soil strength and an increased risk of structural failure. Additionally, fiber agglomeration hinders the even distribution and diffusion of microorganisms within the soil. This results in a reduced and uneven distribution of the cementing agents produced during microbial stabilization, which affects the interface forces between sand particles and fibers. Consequently, this reduces the uniformity and strength of the stabilized soil ^{[10, 11]}.”

The above information has been added to the revised manuscript.

4. In the Section 3.1, the microbial growth curve is an essential parameter for optimizing MICP treatment. It is recommended to include a growth curve for Bacillus pasteurii under optimal conditions to provide a clearer understanding of bacterial activity over time.

Response: Thank you very much for your suggestion. By determining the growth curve under optimal conditions, the different growth stages of Bacillus pasteurii can be clearly identified. These stages help in understanding the microbial growth behavior under varying conditions. Moreover, the growth curve under optimal conditions can serve as a standard for quality control, ensuring the stability of the cultivation conditions and the reproducibility of the experiments.

The growth curve of Bacillus pasteurii under optimal cultivation conditions has been added to Section 3.1.4. A detailed description of the optimal cultivation conditions and the best growth cycle of Bacillus pasteurii is provided below:

3.1.4 Growth Curve Under Optimal Conditions

Through cultivation experiments under varying conditions of urea concentration, pH, and inoculum volume, the optimal growth conditions for Bacillus pasteurii have been determined: urea concentration of 0.5 mol/L, pH of 9, and inoculum volume of 10%. The growth curve of Bacillus pasteurii under these optimal conditions is shown in Figure 7. As seen in the figure, the growth of Bacillus pasteurii can be divided into three stages.

Firstly, from 0 to 24 hours is the growth phase, during which the OD value of the culture and the urease activity of Bacillus pasteurii increase with time. In this phase, the growth rate of Bacillus pasteurii is at its highest, with cell division and reproduction occurring exponentially, indicating an active metabolic state. From 24 to 48 hours, the OD value and urease activity level off, marking the transition to the stationary phase. As nutrients are consumed and waste products accumulate, the growth rate of Bacillus pasteurii slows down, and a dynamic equilibrium between the rates of growth and death is established. During this phase, the bacteria begin to adjust their metabolic processes in response to environmental changes. After 48 hours, the nutrients in the medium become depleted, environmental conditions deteriorate, and the accumulation of waste products and lack of oxygen accelerate bacterial death. At this point, the death rate exceeds the division rate, and the bacteria enter the decline phase. Consequently, the urease activity of the microbes begins to decrease. The slight increase in OD value during this phase is attributed to the accumulation of dead cells and waste products after the microbial population begins to decline.

Figure 8. Growth curve of Bacillus pasteurii under optimal culture conditions(a) Curve of the OD600 value of Bacillus pasteurii. (b) Curve of urease activity of Bacillus pasteurii.

The above information has been added to the revised manuscript.

 

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No comments

Reviewer 3 Report

Comments and Suggestions for Authors

Thanks to the author's time and effort, I think the revised manuscript is acceptable for publication.