Exploiting 1,3-Propanediol Production by a Clostridium beijerinckii Strain: The Role of Glycerol and Ammonium Sulfate Concentrations

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors describe the production of 1,3-PDO from glycerol. The work is interesting.

Minor comments below.

Inorganic species (NH 4 + and SO 4 2- – in the Reviewer's opinion, the choice of words "species" is incorrect. Species is about biology. Please change.

Line 212 – 221

Was the increased glycerol consumption associated with the increased production of 1,3-PDO?

Since the Authors observed a correlation between the amount of glycerol and ammonium sulfate supplementation, is it possible to represent this relationship mathematically?

Can the symbols g and s in equation 1 be changed to other ones? In the Reviewer's opinion, they are associated with the acceleration of gravity and the second (time).

What could have caused the different amounts of butyric acid?Could the Authors explain this further?Is it a different metabolic process or just its speed?

What could have caused the longer lag phase and different glycerol consumption in the two experimental variants?

Line 318 -329

In the Reviewer's opinion, this explanation would be more likely if the number of microorganisms was constant. Were the microorganisms not reproducing (increasing biomass or cell count) during the experiment? Was this an stationary phase?

Author Response

Comment 1: inorganic species (NH 4 + and SO 4 2- – in the Reviewer's opinion, the choice of words "species" is incorrect. Species is about biology. Please change.

Response 1: We have changed species by ions.

 

Comment 2: Line 212 – 221. Was the increased glycerol consumption associated with the increased production of 1,3-PDO?

Response 2: the increase of glycerol consumption was associated with the increase of bacterial growth and consequently 1,3-PDO. We added in Line 221: Thus, increasing glycerol consumption, 1,3-PDO concentration was also increased.

 

Comment 3: Since the Authors observed a correlation between the amount of glycerol and ammonium sulfate supplementation, is it possible to represent this relationship mathematically?

Response 3: Actually, the dependence of glycerol and ammonium sulfate concentrations is already described in the Equation 1: 1,3-PDO (mmol L^-1) = 39,72 + 15,47 s – 7,20 s² + 11,12 n²

Where s is the glycerol (substrate) concentration (mmol L^-1) and n represents the (NH₄)₂SO₄ concentration (mmol L^-1).

 

Comment 4: Can the symbols g and s in equation 1 be changed to other ones? In the Reviewer's opinion, they are associated with the acceleration of gravity and the second (time).

Response 4: g was for glycerol, but we have changed for s (substrate) and n (for nutrient), as described above.

 

Comment 5: What could have caused the different amounts of butyric acid? Could the Authors explain this further? Is it a different metabolic process or just its speed?

Response 5: We have added the following sentence to the manuscript in Line 299.

The difference between butyric acid concentrations (Figure 2E) is probably also due to the higher bacterial growth in the optimized medium. We explained this up to line 299.

.

Comment 6: What could have caused the longer lag phase and different glycerol consumption in the two experimental variants?

Response 6: The addition of ammonium sulfate to the optimized medium leads to improved bacterial growth (Figure 1A) and prolongs pH stability (Figure 2B). As a consequence, in the WISmod optimized medium, both 1,3-PDO and butyric acid concentrations are elevated (Figure 2E). It is likely that the elevated glycerol concentration in the WISmod optimized medium delays the onset of 1,3-PDO production (Figure 2D). However, at the optimal concentration, ammonium sulfate supplementation facilitates higher glycerol consumption relative to WISmod (Figure 2C), ultimately resulting in a higher 1,3-PDO concentration.

 

Comment 7: Line 318 -329: In the Reviewer's opinion, this explanation would be more likely if the number of microorganisms was constant. Were the microorganisms not reproducing (increasing biomass or cell count) during the experiment? Was this an stationary phase?

Response 7: Yes, you are right. However, the cell concentration at the beginning of the assay was similar, but during the assay we could not control it. The higher bacterial growth was exactly the effect of the optimized medium.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript describes the effect of glycerol and (NH₄)₂SO₄ concentrations on 1,3-PDO production and cell growth in Closteridium beijerinckii strain Br21. The authors used a central composite rotational design and statistical significance to determine optimal glycerol and (NH₄)₂SO₄ concentrations and demonstrated increased 1,3-PDO production compared with the original medium. They also reported the effect of the optimized medium on expression of several genes involved in 1,3-PDO production from glycerol. Overall, this is a novel study that will be of interest to those who are investigating glycerol fermentation. However, there are several issues that the authors should address:

1. Line 94- Where is the sequence deposited? Please clarify (such as GenBank accession number MWMH00000000).
2. Methods section- I see conditions for growing the inoculum and for medium preparation in 100 mL flasks (presumably for the growth tests), but I do not see any conditions for the “kinetic assays”. Were these kinetic assays performed in growth medium? What were the temperature and agitation rates for the assays?
3. Lines 192-195- there is an extra half sentence (repeating the end of the previous sentence) that should be removed
4. Where the authors refer to OD (lines 199, 207, Table 2, etc.), they should instead use OD₆₀₀ to indicate at which wavelength the OD was measured.
5. Table 2 legend- please describe what the numbers in parentheses under glycerol and (NH₄)₂SO₄
6. Line 212- “ranging 51.36” should say “ranging from 51.36”
7. Lines 229-231- the authors claim that increasing (NH₄)₂SO₄ concentrations will give higher glycerol consumption, citing conditions 1 vs. 2 and 7 vs. 8. However, conditions 9 and 10 appear to have a moderate amount of glycerol (more than condition 7, less than condition 8) and yet they both have a lower glycerol consumption rate. Also, what is the difference between conditions 9 and 10? They appear to be the same.
8. Lines 251-255- the authors claim that the optimal conditions for 1,3-PDO production are 441.42 mmol/L glycerol and 25.8 mmol/L (NH₄)₂SO₄, but those are the maximum points on the graph. Why did the authors not test higher concentrations to see if the maximum point on the graph in Fig. 1 is a true maximum?
9. Figure 2E- why does the butyric acid concentration decrease toward the end of the fermentation? Please provide a brief explanation.
10. Lines 363-365- the authors mention that the optimized medium “did not result in a significant alteration of the expression of the gene that encodes the glycerol uptake protein.” Is this not the same as the description of glpF expression in lines 356-357? Please delete one of the two references to avoid repeating the same results.
11. Lines 378-380- the authors state that the gene upregulation observed supports the hypothesis that 1,3-PDO production is enhanced in the optimal medium. The gene expression doesn’t really support the fact observation that the optimal medium results in higher 1,3-PDO production- the fermentation results in Fig. 2 do that well enough. Instead, the gene upregulation data provides an explanation for why the 1,3-PDO production is increased by looking at what enzymes are present.

Author Response

Reviewer 2

The manuscript describes the effect of glycerol and (NH₄)₂SO₄ concentrations on 1,3-PDO production and cell growth in Clostridium beijerinckii strain Br21. The authors used a central composite rotational design and statistical significance to determine optimal glycerol and (NH₄)₂SO₄ concentrations and demonstrated increased 1,3-PDO production compared with the original medium. They also reported the effect of the optimized medium on expression of several genes involved in 1,3-PDO production from glycerol. Overall, this is a novel study that will be of interest to those who are investigating glycerol fermentation. However, there are several issues that the authors should address:

 

 

1. Line 94- Where is the sequence deposited? Please clarify (such as GenBank accession number MWMH00000000).

Response 1: Yes, we do have a deposited sequence. Sorry the GenBank number: MWMH01000003.1. WE have corrected this in the manuscript.

Comment 2: Methods section- I see conditions for growing the inoculum and for medium preparation in 100 mL flasks (presumably for the growth tests), but I do not see any conditions for the “kinetic assays”. Were these kinetic assays performed in growth medium? What were the temperature and agitation rates for the assays?

Response 2: you are right, we have detailed this method in the manuscript up the line 119.

Kinetic assays were performed using both culture media, the WISMod medium (section 2.2) and the medium under optimized glycerol and (NH₄)₂SO₄ concentrations, according the results of CCRD. The inoculum was made by introducing a volume of the pre-inoculum to give 0.1 of optical density at 600 nm at the beginning of the assays. After the inoculum the flasks were maintained at 33°C with agitation at 150 rpm for 192 hours.

 

Comment 3: Lines 192-195- there is an extra half sentence (repeating the end of the previous sentence) that should be removed

Response 3: Thank you, it was removed.

 

Comment 4: Where the authors refer to OD (lines 199, 207, Table 2, etc.), they should instead use OD₆₀₀ to indicate at which wavelength the OD was measured.

Response 4: Thank you, we have performed the changes.

 

Comment 5: Table 2 legend- please describe what the numbers in parentheses under glycerol and (NH₄)₂SO₄

Response 5: We have changed the table legend to include this information.

Table 2. – Conditions used in the experiments indicated by the CCRD and results of final pH and OD600, final concentrations of butyric acid, 1,3-PDO, and consumed glycerol. Values are represented as the average obtained from biological triplicate fermentations, with errors calculated from the standard deviation. The values in parentheses, present in the conditions, represent the coded levels of the variables, which were used to obtain the model.

 

Comment 6: Line 212- “ranging 51.36” should say “ranging from 51.36”

Response 6: OK, thank you for your help.

 

Comment 7: Lines 229-231- the authors claim that increasing (NH₄)₂SO₄ concentrations will give higher glycerol consumption, citing conditions 1 vs. 2 and 7 vs. 8. However, conditions 9 and 10 appear to have a moderate amount of glycerol (more than condition 7, less than condition 8) and yet they both have a lower glycerol consumption rate. Also, what is the difference between conditions 9 and 10? They appear to be the same.

Response 7:  Yes, 9 and 10 are the central points of this methodology that needs to be repeated to validated the model. We have included the following explanation in the manuscript (line 227 to 237):

Assays 9 and 10 exhibited equivalent glycerol concentrations to assays 7 and 8, but a reduced ammonium sulfate concentration compared to assay 8. Consequently, assays 9 and 10 demonstrated a lower glycerol consumption rate than assay 8. Conversely, assay 7 which had the same glycerol concentration and a lower ammonium sulfate concentration, resulted in a higher glycerol consumption rate than assays 9 and 10. This trend is corroborated by the response surface depicted in Figure 1, suggesting a potential antagonistic interaction between the variables, wherein a median ammonium sulfate concentration appears to be suboptimal. Such non-linear behavior is frequently observed in biological assays, justifying the selection of a Central Composite Rotatable Design (CCRD) optimization methodology. The CCRD approach minimizes experimental errors and facilitates the determination of an optimal concentration range.

 

Comment 8: Lines 251-255- the authors claim that the optimal conditions for 1,3-PDO production are 441.42 mmol/L glycerol and 25.8 mmol/L (NH₄)₂SO₄, but those are the maximum points on the graph. Why did the authors not test higher concentrations to see if the maximum point on the graph in Fig. 1 is a true maximum?

Response 8: You are right, we could have tested higher concentrations, but the graphical representation also shows that we reached the maximum in the range we have analyzed. In the CCRD methodology, when aiming to maximize a response, the optimal point is determined as the highest response among the evaluated points. Furthermore, regarding glycerol concentration, a plateau is observed at higher concentrations, indicating that increasing it might not be beneficial. However, increasing the ammonium sulfate concentration could indeed have been further investigated.

 

 

Comment 9: Figure 2E- why does the butyric acid concentration decrease toward the end of the fermentation? Please provide a brief explanation.

Response 9: Line 324

Notably, butyric acid concentration declined at the end of the kinetic assays (Figure 2E). This suggests its potential utilization by C. beijerinckii as an electron acceptor for sulfate reduction, given the continued decrease in sulfate concentration observed at the assay's endpoint.

 

Comment 10: Lines 363-365- the authors mention that the optimized medium “did not result in a significant alteration of the expression of the gene that encodes the glycerol uptake protein.” Is this not the same as the description of glpF expression in lines 356-357? Please delete one of the two references to avoid repeating the same results.

Response 10: Thank you, we have done this.

 

Comment 11: Lines 378-380- the authors state that the gene upregulation observed supports the hypothesis that 1,3-PDO production is enhanced in the optimal medium. The gene expression doesn’t really support the fact observation that the optimal medium results in higher 1,3-PDO production- the fermentation results in Fig. 2 do that well enough. Instead, the gene upregulation data provides an explanation for why the 1,3-PDO production is increased by looking at what enzymes are present.

Response 11: Line 406: Thus, the increased 1,3-PDO production can be explained at a molecular level by the upregulation of these genes, which leads to a corresponding increase in the expression of the involved genes and probably in the activities of the enzymes.