Microbial Protein Production Using Lignocellulosic Biomass (Switchgrass) and Klebsiella oxytoca M5A1—A Nitrogen Fixer

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The reviewer has already reviewed this manuscript and the authors have made the necessary change to consider for publication. Good work by all authors.

Author Response

Comment: The reviewer has already reviewed this manuscript and the authors have made the necessary

Response: Thanks

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The authors have fully addressed my comments and the overall quality of this manuscript is significantly improved.  

Author Response

Comment 1: The authors have fully addressed my comments and the overall quality of this manuscript is.
Response: Thanks

Reviewer 3 Report (Previous Reviewer 4)

Comments and Suggestions for Authors

In the first round of responses, the author stated: "Equivalent of 10g/L of the hydrolysate was used for the fermentation medium. The highest protein concentration observed was 683.46 microgram/mL, which is approximately 68.3% if calculated in percentage". However, microorganisms are unable to convert 10 g/L of substrate (hydrolysate) into 683.46 mg/mL products (protein). The product far exceeds the substrate, which goes against basic common sense.

Khumchai et al. reported the total crude protein (SCP) as 5.3 g L⁻¹ (78.79%) in 30 days of bioremediation process. The total crude protein was 5.3 g/L, not 787.9 g/L. The 78.79% refers to the dried biomass of S. tuirus OS1 containing 78.79% of total protein as SCP.

"Khumchai, J., Wongchai, A., On-uma, R., Sabour, A., Alshiekheid, M., Narayanan, M., Karuppusamy, I., Pugazhendhi, A., Brindhadevi, K., & Lan Chi, N. T. (2022). A viable bioremediation strategy for treating paper and pulp industry effluents and assessing the prospect of resulted bacterial biomass as single cell protein (SCP) using indigenous bacterial species. Chemosphere, 304, 135246. https://doi.org/10.1016/j.chemosphere.2022.135246 "

As shown in Fig. 3, the microbial protein concentration was lower than 700 ug/mL, which was much lower than 683.46 mg/mL.

 

The nitrogen fixation ability of Klebsiella oxytoca M5A1 should be experimentally proven in this study. Otherwise, nitrogen fixation should not appear in the title.

 

The current manuscript does not provide meaningful results and the experiment is too simple. The author needs to supplement more experiments to make this study meaningful.

Author Response

Comment 1: In the first round of responses, the author stated: "Equivalent of 10g/L of the hydrolysate was used for the fermentation medium. The highest protein concentration observed was 683.46 microgram/mL, which is approximately 68.3% if calculated in percentage". However, microorganisms are unable to convert 10 g/L of substrate (hydrolysate) into 683.46 mg/mL products (protein). The product far exceeds the substrate, which goes against basic common sense. Khumchai et al. reported the total crude protein (SCP) as 5.3 g L−1 (78.79%) in 30 days of bioremediation process. The total crude protein was 5.3 g/L, not 787.9 g/L. The 78.79% refers to the dried biomass of S. tuirus OS1 containing 78.79% of total protein as SCP. "Khumchai, J., Wongchai, A., On-uma, R., Sabour, A., Alshiekheid, M., Narayanan, M., Karuppusamy, I., Pugazhendhi, A., Brindhadevi, K., & Lan Chi, N. T. (2022). A viable bioremediation strategy for treating paper and pulp industry effluents and assessing the prospect of resulted bacterial biomass as single cell protein (SCP) using indigenous bacterial species. Chemosphere, 304, 135246. https://doi.org/10.1016/j.chemosphere.2022.135246 ". As shown in Fig. 3, the microbial protein concentration was lower than 700 ug/mL, which was much lower than 683.46 mg/mL.

Response 1: Thank you for the observation. The percentage was miscalculated. The product concentration was 683.46 microgram/mL.

Comment 2: The nitrogen fixation ability of Klebsiella oxytoca M5A1 should be experimentally proven in this study. Otherwise, nitrogen fixation should not appear in the title.

Response 2:  As suggested, we have experimentally confirmed their nitrogen fixation ability and the methodology and result are presented in the revision. Line 166 - 179

Comment 3: The current manuscript does not provide meaningful results and the experiment is too simple. The author needs to supplement more experiments to make this study meaningful.

Response 3: Done. We have added a comparison data of microbial protein production using conventional sucrose as carbon source. We have also carried out Acetylene reduction assay to experimentally confirm Nitrogen fixation ability of K. oxytoca M5A1.

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

The article sustainability-3020986 is devoted to the production of microbial protein from Switchgrass, with nitrogen-fixing bacteria used as a protein producer. The work was carried out at a good methodological level. The use of pre-treatment with aqueous ammonia and enzymatic hydrolysis with Cellic HTec appears to be justified. Interesting data were obtained on the profile of organic acids produced by Klebsiella oxytoca M5A1 and their metabolism.

The article can be improved; to do this, it is necessary to eliminate a number of shortcomings and answer questions.

1) The title does not reflect the essence of the work; it is necessary to specifically write what lignocellulosic biomass was used and what nitrogen-fixing producer was used.

2) The authors indicate that the Klebsiella oxytoca M5A1 producer used is non-toxic. However, this is the first time that microbial protein has been obtained using this producer, which is the novelty of the work. Therefore, it is necessary to confirm that the microbial protein is non-toxic. It is known that sometimes, in parallel with protein synthesis, the synthesis of toxins occurs; in this case, it is necessary to confirm the absence of toxins.

3) Line 150, line 188. It is necessary to justify why such a low substrate concentration of 10 g/l is used. If the initial concentration of the substrate was 30 g/l, then after 72 hours getting 10 g/l of glucose is very little. Such a process will not be cost-effective. It may be necessary to change the pretreatment conditions to obtain a more fermentable substrate.

4) Figure 1. The maximum OD absorption is observed after 48 hours, then it seems that after 56, 64, 72 hours the OD will continue to increase. Why was the experiment interrupted? It has not been proven that 48 hours is the extreme point.

5) Line 297 and line 304 according to Figure 3, it is better to write “between 8 and 24 hours” rather than “between 16 and 24 hours”.

6) It’s a pity that the authors only took into account changes in glucose concentration; perhaps Klebsiella oxytoca M5A1 also utilizes other sugars. It was possible to take into account changes in glucose concentration and changes in the concentration of reducing substances in general.

Author Response

Comment 1:  The title does not reflect the essence of the work; it is necessary to specifically write what lignocellulosic biomass was used and what nitrogen-fixing producer was used.

Response 1: Done. The title has been adjusted. The aim of this study initially is to prove that K.oxytoca M5A1 can use lignocelluloic hydrolysate for growth and product formation. However, we have experimentally confirmed their nitrogen fixation ability. The methodology and result are presented in Line 166 - 179 and 373 – 305 respectively.

Comment 2: The authors indicate that the Klebsiella oxytoca M5A1 producer used is non-toxic. However, this is the first time that microbial protein has been obtained using this producer, which is the novelty of the work. Therefore, it is necessary to confirm that the microbial protein is non-toxic. It is known that sometimes, in parallel with protein synthesis, the synthesis of toxins occurs; in this case, it is necessary to confirm the absence of toxins.

Response 2: Thank you for the observation. It is beyond the scope of this project, however, it is worth considering for future research endeavors. This has been included in conclusion.

Comment 3: Line 150, line 188. It is necessary to justify why such a low substrate concentration of 10 g/l is used. If the initial concentration of the substrate was 30 g/l, then after 72 hours getting 10 g/l of glucose is very little. Such a process will not be cost-effective. It may be necessary to change the pretreatment conditions to obtain a more fermentable substrate.

Response 3: The enzymatic hydrolysis process yielded above 20g/L substrate concentration. We only did the dilution to have approximately 10g/L concentration for fermentation. This is to observe the performance of the microbe in this amount of substrate, with the aim of scaling up as the experiment progresses.

Comment 4: Figure 1. The maximum OD absorption is observed after 48 hours, then it seems that after 56, 64, 72 hours the OD will continue to increase. Why was the experiment interrupted? It has not been proven that 48 hours is the extreme point.

Response 4: As much as we wanted to prove the K.oxytoca M5A1 can grow in switch hydrolysate, we are also very much interested in the product formation. At 48 h, the production of MP began to drop which can be as a result of substrate depletion. Additionally, during our preliminary experiment, we observed that at 72 h the growth of the microbe had decreased (which can also be explained by depletion in substrate concentration).

Comment 5: Line 297 and line 304 according to Figure 3, it is better to write “between 8 and 24 hours” rather than “between 16 and 24 hours”.

Response 5: Done. Thank you for your suggestion.

Comment 6: It’s a pity that the authors only took into account changes in glucose concentration; perhaps Klebsiella oxytoca M5A1 also utilizes other sugars. It was possible to take into account changes in glucose concentration and changes in the concentration of reducing substances in general.

Response 6: Yes, it is possible. However, we took into account changes in glucose because it is the most abundant sugar in our substrate.

Round 2

Reviewer 3 Report (Previous Reviewer 4)

Comments and Suggestions for Authors

The manuscript can be accepted.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript sustainability-2970246 by Ayodele et al. describes a novel strategy of microbial protein production using lignocellulosic biomass as economic carbon source and nitrogen fixation ability of K. oxytoca M5A1 as nitrogen source. The manuscript characterized K. oxytoca growth in the presence of LCB in multiple perspective including glucose consumption, protein production at different tested time points, and yield of side products such as organic acids. Authors claim that combination of LCB and K. oxytoca M5A1 is an eco-fridendly and cost-effective way to alternate traditional protein sources. 

This is a well-written paper and I only have minor comments:

Figures: it is recommended that authors add figure legends and captions for all the figures. It's very hard to understand each figure without a description of techniques, explanation of features in each figure, calculation of error bar, and difference in values at different time points statistically different or not?

Section 2.5 in line 129: authors only mention about the samples were analyzed by HPLC and results were reported in g/L. It's recommended that authors to add description of how analytes were been quantified by HPLC, e.g. did you use internal standards for glucose and other organic acids mentioned in Section 3.4.

Reviewer 2 Report

Comments and Suggestions for Authors

The work’s goal was to produce protein using lignocellulosic biomass (LCB) as a carbon source and the nitrogen fixation ability of Klebsiella oxytoca M5A1 as a nitrogen source. The authors report protein production peaked at 40 hr by measuring concentration using Bradford assay (Figure 3). The study demonstrates utilization of LCB and nitrogen fixing bacterial strain (M5A1) could be useful for microbial-based protein production that is cost-effective alternate to traditional process. The manuscript doesn’t add any significant improvement in the field of utilization of microorganism in protein production. No clear reasoning is mentioned about the use of M5A1 strain and how the protein source from the fermentation process can be further utilized for food manufacturing. The figure legends and results section need extensive work and more description on the interpretation of the results. A detailed study on the cost-benefit analysis compared to other methods would have also helped improve the manuscript’s impact. Overall, the manuscript needs more data and improved hypothesis

Reviewer 3 Report

Comments and Suggestions for Authors

This paper reported a method to generate protein using lignocellulosic biomass as a carbon source and the nitrogen fixation ability of Klebsiella oxytoca M5A1 as a nitrogen source. The protein and acid concentration are reported at different times. However, the experimental results are not properly validated, and the quality of figures and organization of writing should be improved. This paper proposes to utilize the nitrogen-fixing bacteria to provide the source of nitrogen for protein production, but the efficiency of this method and how it compared to the previous methods are not well demonstrated.

 

1.      The samples were taken every 8 hours and the concentration of glucose (such as in figure 2) was estimated. How did the author make sure the profile correctly represents the actual utilization rate of glucose? Could the profile be different if the sample were taken out every 1 hour? Same issues go to figure1, figure 3 and figure 4.

 

2.      In section 3.3, paragraph 3, line 258-271, the authors summarize the previous research on the utilization of various substrates for microbial protein production. Should this paragraph go to the introduction section?  In the end of this paragraph, “The variations observed in these studies confirm that the yield of microbial protein produced during fermentation is not only influenced by the type of substrate used but also microbial strain employed”. It would be much better if the authors list out the protein content in different studies and try to connect the variation in results with the substrate and microbial strain instead of making a conclusion directly.

 

3.      In Section 3.4 , “Phosriran et al. [27] reported that an engineered 315 strain of K. oxytoca M5A1 (KMS006) produced SA at a concentration of 4.82 g/L, while 316 the wild-type K. oxytoca M5A1 strain produce 1.47g/L SA. Although our study observed a concentration of 1.2 g/L of SA, the yield of 1.47 g/L by the wild-type strain in Phosriran et al.'s study aligns closely with our findings, suggesting consistency between our results and those of the wild-type strain.”  The authors should add how they obtain the concentration “1.2 g/L of SA” and explain the reason that caused the difference, instead of simply stating the results closely aligned with others’ findings.

 

4.      In the conclusion paragraph, “Meaning that the strain is able to use a cheaper source of carbon while harnessing free 359 Sustainability 2024, 16, x FOR PEER REVIEW 10 of 11  nitrogen from the atmosphere.” Is a wrongly written sentence. Although the underlying mechanism is mentioned to be fugure down in the future work, the explanation and justification for the trend of the observed dynamic trend should be added.

Comments on the Quality of English Language

Some of the paragraphs are not well organized, and one sentence was found to be grammarly wrong, as mentioned in comments. The authors should review and double check the whole paper to make sure the ideas are clearly expressed. 

Reviewer 4 Report

Comments and Suggestions for Authors

In this study, microbial proteins were produced using lignocellulosic biomass as raw material through Klebsiella oxytoca. The growth proffle, glucose utilization, protein and organic acid formation have been studied during the fermentation of K. oxytoca. However, the focus of this study is not prominent, the experimental design is too simple, and the analysis data is also limited.

 

1. The author found that the highest protein concentration was 68.3%. How many substrates were converted to achieve such a high protein concentration? Is the result of protein concentration measurement accurate?

 

2. What are the types of microbial proteins produced by Klebsiella oxytoca M5A1?

 

3. Can the nitrogen fixation ability of Klebsiella oxytoca M5A1 be experimentally proven? What is its metabolic mechanism?